Azacitidine in combination with venetoclax, gilteritinib, midostaurin or other compounds for treating leukemia or myelodysplastic syndrome

ABSTRACT

Provided herein are methods for using 5-azacytidine in combination with one or more additional agents to treat diseases and disorders including acute myeloid or myelogenous leukemia (AML). Other disease and disorders those associated with abnormal cell proliferation, myelodysplastic syndromes (MDS), abnormal cell proliferation, hematologic disorders, and immune disorders, among others.

RELATED APPLICATIONS

This application claims the benefits of priority of U.S. provisionalapplication 62/864,413 filed Jun. 20, 2019, the entire contents of whichare incorporated herein by reference.

FIELD

Provided are methods for using 5-azacytidine in combination with one ormore additional therapeutic agents to treat diseases and disordersincluding cancers such as but not limited to acute myeloid leukemia(AML), myelodysplastic syndromes (MDS), disorders related to abnormalcell proliferation, hematologic disorders, and immune disorders, amongothers.

BACKGROUND

Cancer is a major worldwide public health problem with many types ofcancer that have been described in the medical literature. Examplesinclude cancer of the blood, bone, lung (e.g., non-small-cell lungcancer and small-cell lung cancer), colon, breast, prostate, ovary,brain, and intestine. The incidence of cancer continues to climb as thegeneral population ages and as new forms of cancer develop. A continuingneed exists for effective therapies to treat cancer patients.

Myelodysplastic syndromes (MDS) refers to a diverse group ofhematopoietic stem cell disorders. The annual incidence of MDS isestimated to be 4.9 cases per 100,000 people worldwide, andapproximately 10,000 people in the United States are diagnosed with MDSeach year. MDS may be characterized by a cellular marrow with impairedmorphology and maturation (dysmyelopoiesis), peripheral bloodcytopenias, and a variable risk of progression to acute leukemia,resulting from ineffective blood cell production.

MDS are grouped together because of the presence of dysplastic changesin one or more of the hematopoietic lineages including dysplasticchanges in the myeloid, erythroid, and megakaryocytic series. Thesechanges result in cytopenias in one or more of the three lineages.Patients afflicted with MDS may develop complications related to anemia,neutropenia (infections), and/or thrombocytopenia (bleeding). About 10%to about 70% of patients with MDS may develop acute leukemia. In theearly stages of MDS, the main cause of cytopenias is increasedprogrammed cell death (apoptosis). As the disease progresses andconverts into leukemia, a proliferation of leukemic cells overwhelms thehealthy marrow. The disease course differs, with some cases behaving asan indolent disease and others behaving aggressively with a very shortclinical course that converts into an acute form of leukemia. Themajority of people with higher risk MDS eventually experience bonemarrow failure. Up to 50% of MDS patients succumb to complications, suchas infection or bleeding, before progressing to AML.

Acute myeloid leukemia (AML) is a type of cancer that affects the bonemarrow and blood. AML is known by a variety of names, including acutemyelogenous leukemia, acute myeloblastic leukemia, acute granulocyticleukemia, and acute nonlymphocytytic leukemia. The word “acute” in acutemyelogenous leukemia reflects the disease's rapid progression. It iscalled acute myeloid leukemia because it affects a group of white bloodcells called the myeloid cells, which normally develops into the varioustypes of mature blood cells, such as red blood cells, white blood cells,and platelets. In other words, AML is a malignancy of the myeloidprecursor cell line, characterized by the rapid proliferation ofabnormal cells, which accumulate in the bone marrow and interfere withthe production of normal cells.

AML is generally classified as de novo, or secondary when arisingfollowing exposure to prior cytotoxic chemotherapy, or after a historyof prior myelodysplastic syndrome (MDS) or antecedent hematologicdisorder (AHD). The pathogenesis of AML at the genetic level is alsoheterogeneous. Genetic alterations that cause AML include an internaltandem duplication in a tyrosine kinase gene, chromosomal rearrangementsthat alter the functioning of genes involved in leukemogenesis, andmutations resulting in activation of transcription factors, etc.Comprehensive profiling of genetic alterations in AML will enhancedisease classification, risk stratification and prognosis, andultimately, allow more precise therapeutic interventions. MV4-11 andMOLM-13 are AML cell lines that express FLT3 mutations. See Quentmeieret al., Leukemia, 17(1):120-4 (January 2003). FLT3-ITD up-regulatesMCL-1 to promote survival of stem cells in AML. See Yoshimoto et al.,Blood, 114(24):5034-43 (Dec. 3, 2009).

Current strategies of AML treatment include inductive chemotherapy forremission induction and low-intensity therapy intended for survivalprolongation. The remission-induction chemotherapy is a cytoreductivemodality for achieving remission or at least effective reduction oftumor burden. The combination of cytarabine and anthracycline has beenthe mainstay of treatments to induce remission. A common inductionregimen consists of cytarabine at doses of 100 to 200 mg/m²/day for 7days and daunorubicin at doses of 45 to 90 mg/m²/day for 3 days, oftenreferred to as the “7+3 protocol.” If remission is achieved, additionalcycles of chemotherapy or stem cell transplantation from a donor(allogeneic hematopoietic stem cell transplantation [HSCT]) are employedfor consolidation. Although inductive chemotherapy has become thestandard for younger fit patients, it remains a matter of debate in theelderly and unfit population. In elderly patients who have received IC,outcomes are less favorable primarily due to the increased rate oftreatment-related death and poor prognostic factors leading to lowerremission rates seen in the elderly population. Treatment options forpatients considered ineligible or unfit due to age, performance status,and co-morbidities or those who choose not to receive IC currentchemotherapy options include low-dose cytarabine, 5-azacytidine ordecitabine.

Although induction chemotherapy produces morphologic complete remissions(CRs) in about 60% to 80% of younger adults and 40% to 50% of olderadults with newly diagnosed AML, there is a substantial population ofpatients who will fail to attain CR (i.e., refractory AML). Even forthose who attain CR after induction treatment, a significant portionwill eventually relapse, leading to only about 29% relapse-free survivalat three years after treatment.

Thus, there is a need for more effective treatments for treating cancer,including but not limited to AML and/or MDS, and this disclosureaddresses this need.

SUMMARY

Provided herein are methods of treating diseases and disorders includingAML, MDS, cancer, disorders related to abnormal cell proliferation,hematologic disorders, and immune disorders using 5-azacytidine incombination with at least one additional therapeutic agent.

Certain embodiments herein provide that the additional therapeutic agentis gilteritinib, midostaurin, quizartinib, enasidenib, ivosidenib, orvenetoclax.

Certain embodiments herein provide that the 5-azacytidine isadministered as a composition that is a single unit dosage form. Certainembodiments herein provide compositions that are non-enteric-coated.Certain embodiments herein provide compositions that are immediaterelease oral compositions.

Provided in one aspect is a method of treating a human having acutemyeloid leukemia or myelodysplastic syndrome, wherein the methodcomprises administering to the human (i) a pharmaceutical compositioncomprising 5-azacytidine administered orally, and (ii) at least oneadditional therapeutic agent.

In some embodiments, the at least one additional therapeutic agentcomprises a FMS-like tyrosine kinase 3 (FLT3) inhibitor, an isocitratedehydrogenase 2 (IDH2) inhibitor, an isocitrate dehydrogenase 2 (IDH1)inhibitor, and/or a B-cell lymphoma 2 (BCL2) inhibitor.

In some embodiments, the at least one additional therapeutic agentcomprises gilteritinib, midostaurin, quizartinib, enasidenib,ivosidenib, and/or venetoclax. In some embodiments, the at least oneadditional therapeutic agent is gilteritinib or midostaurin. In someembodiments, the at least one additional therapeutic agent isgilteritinib. In some embodiments, the at least one additionaltherapeutic agent is venetoclax.

In some embodiments, the 5-azacytidine and the at least one additionaltherapeutic agent are administered concomitantly. In some embodiments,the 5-azacytidine and the at least one additional therapeutic agent areadministered sequentially wherein the 5-azacytidine is administeredfirst.

In some embodiments, the 5-azacytidine and the at least one additionaltherapeutic agent are co-formulated as a single unit dosage form. Insome embodiments, the 5-azacytidine and the at least one additionaltherapeutic agent are formulated as separate dosage forms.

In some embodiments, the at least one additional therapeutic agent isadministered parenterally. In some embodiments, the at least oneadditional therapeutic agent is administered orally.

In some embodiments, the 5-azacytidine is administered at a dose ofabout 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, about250 mg, about 260 mg, about 270 mg, about 280 mg, about 290 mg, about300 mg, about 310 mg, about 320 mg, about 330 mg, about 340 mg, about350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, about400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg, about450 mg, about 460 mg, about 470 mg, about 480 mg, about 490 mg, about500 mg, about 510 mg, about 520 mg, about 530 mg, about 540 mg, about550 mg, about 560 mg, about 570 mg, about 580 mg, about 590 mg, or about600 mg orally. In some embodiments, the 5-azacytidine is administered ata dose of about 200 mg. In some embodiments, the 5-azacytidine isadministered at a dose of about 300 mg. In some embodiments, the5-azacytidine is administered at a dose of 50 mg, 60 mg, 70 mg, 80 mg,90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg,180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg,270 mg, 280 mg, 290 mg, 300 mg, 310 mg, 320 mg, 330 mg, 340 mg, 350 mg,360 mg, 370 mg, 380 mg, 390 mg, 400 mg, 410 mg, 420 mg, 430 mg, 440 mg,450 mg, 460 mg, 470 mg, 480 mg, 490 mg, 500 mg, 510 mg, 520 mg, 530 mg,540 mg, 550 mg, 560 mg, 570 mg, 580 mg or 600 mg orally. In someembodiments, the 5-azacytidine is administered at a dose of 200 to 300mg orally. In some embodiments, the 5-azacytidine is administered at adose of 200 mg. In some embodiments, the 5-azacytidine is administeredat a dose of 300 mg. In some embodiments, the 5-azacytidine isadministered daily for the first seven, fourteen, or twenty-one days ofa 28-day cycle. In some embodiments, the 5-azacytidine is administeredto the human one or two times per day. In some embodiments, the5-azacytidine is administered to the human once per day. In someembodiments, the 5-azacytidine is administered in the form of a capsuleor a tablet. In some embodiments, the 5-azacytidine is administered inthe form of a non-enteric-coated tablet. In some embodiments, the5-azacytidine is administered in the form of an immediate release oralcomposition.

In some embodiments, the 5-azacytidine is administered at a dose ofabout 200 mg per day for 14 days in a 28-day cycle. In some embodiments,the 5-azacytidine is administered at a dose of about 300 mg per day for14 days in a 28-day cycle. In some embodiments, the 5-azacytidine isadministered at a dose of about 200 mg per day for 21 days in a 28-daycycle. In some embodiments, the 5-azacytidine is administered at a doseof about 300 mg per day for 21 days in a 28-day cycle. In someembodiments, the 5-azacytidine is administered at a dose of about 200 mgper day for 7 days in a 28-day cycle. In some embodiments, the5-azacytidine is administered at a dose of about 300 mg per day for 7days in a 28-day cycle. In some embodiments, the 5-azacytidine isadministered at a dose of 200 mg per day for 14 days in a 28-day cycle.In some embodiments, the 5-azacytidine is administered at a dose of 300mg per day for 14 days in a 28-day cycle. In some embodiments, the5-azacytidine is administered at a dose of 200 mg per day for 21 days ina 28-day cycle. In some embodiments, the 5-azacytidine is administeredat a dose of 300 mg per day for 21 days in a 28-day cycle. In someembodiments, the 5-azacytidine is administered at a dose of 200 mg perday for 7 days in a 28-day cycle. In some embodiments, the 5-azacytidineis administered at a dose of 300 mg per day for 7 days in a 28-daycycle.

In some embodiments, the 5-azacytidine is administered (a) daily for 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or greater than 14 days,optionally followed by a treatment dosing holiday of 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, or greater than 14 days; (b) daily for 14or more days, optionally followed by a treatment dosing holiday of 7 ormore days; (c) for 21 or more days, optionally followed by a treatmentdosing holiday of 7 or more days; (d) for 14 days, optionally followedby a treatment dosing holiday of 14 days; (e) for 21 or more days,followed by a treatment dosing holiday of 7 or more days; (f) for 14days, followed by a treatment dosing holiday of 14 days. In someembodiments, at least one of steps (a), (b), (c), (d), (e), or (f) arerepeated.

In some embodiments, the 5-azacytidine is administered (a) at a dose ofabout 300 mg daily for 14 days, followed by a treatment dosing holidayof 14 days; (b) at a dose of about 200 mg daily for 14 days, followed bya treatment dosing holiday of 14 days; (c) at a dose of about 300 mgdaily for 21 days, followed by a treatment dosing holiday of 7 days; (d)at a dose of about 200 mg daily, followed by a treatment dosing holidayof 7 days; (e) at a dose of 300 mg daily for 14 days, followed by atreatment dosing holiday of 14 days; (f) at a dose of 200 mg daily for14 days, followed by a treatment dosing holiday of 14 days; (g) at adose of 300 mg daily for 21 days, followed by a treatment dosing holidayof 7 days; or (h) at a dose of 200 mg daily, followed by a treatmentdosing holiday of 7 days. In some embodiments, at least one of steps(a), (b), (c), (d), (e), (f), (g), or (h) are repeated.

In some embodiments, the 5-azacytidine is administered using a treatmentcycle comprising administration of 5-azacytidine per day for 7 days in a28-day cycle In some embodiments, the 5-azacytidine is administeredusing a treatment cycle comprising administration of 5-azacytidine perday for 14 days in a 28-day cycle. In some embodiments, the5-azacytidine is administered using a treatment cycle comprisingadministration of 5-azacytidine per day for 21 days in a 28-day cycle.

In some embodiments, the 5-azacytidine and the at least one additionaltherapeutic agent provides a synergistic effect to treat the acutemyeloid leukemia or myelodysplastic syndrome.

In some embodiments, the acute myeloid leukemia is characterized ascaused by a FMS-like tyrosine kinase-3 internal tandem duplication(FLT3-ITD) mutation. In some embodiments, the 5-azacytidine isadministered before the at least one additional therapeutic agent. Insome embodiments, the 5-azacytidine and at least one additionaltherapeutic agent augments myeloid cell leukemia 1 (MCL-1) degradation.

In some embodiments, the 5-azacytidine and at least one additionaltherapeutic agent increases median survival as compared to 5-azacytidineadministered intravenously or subcutaneously and at least one additionaltherapeutic agent. In some embodiments, the 5-azacytidine and at leastone additional therapeutic agent increases median survival as comparedto 5-azacytidine administered intravenously or subcutaneously and atleast one additional therapeutic agent by about 10%, about 15%, about20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%,about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about85%, about 90%, about 95%, or about 100%.

In some embodiments, the acute myeloid leukemia is resistant totreatment with at least one additional therapeutic agent. In someembodiments, the acute myeloid leukemia is responsive to treatment witha FMS-like tyrosine kinase-3 (FLT3 inhibitor). In some embodiments, theacute myeloid leukemia is characterized as having an overexpression ofMCL-1. In some embodiments, the 5-azacytidine primes the cancer cellsfor apoptosis mediated by the at least one additional therapeutic agentby downregulating the expression of MCL-1. In some embodiments, the5-azacytidine and at least one additional therapeutic agent augmentsMCL-1 degradation. In some embodiments, the 5-azacytidine alters cellmetabolism. In some embodiments, the 5-azacytidine causes cell cyclearrest. In some embodiments, the 5-azacytidine suppresses oxidativephosphorylation. In some embodiments, the 5-azacytidine increasesexpression of activating transcription factor 3 (ATF3). In someembodiments, the 5-azacytidine decreases expression of stearoyl-CoAdesaturase (SCD).

In some embodiments, the method comprises: (a) administering the5-azacytidine daily to the human for 1, 2, or 3 days; (b) administeringthe at least one additional therapeutic agent to the human for one ormore days; and (c) optionally repeating steps (a) and (b).

In some embodiments, the method comprises: (a) administering the5-azacytidine daily to the human for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, or 14 days; (b) administering the at least one additionaltherapeutic agent to the human for one or more days; and (c) optionallyrepeating steps (a) and (b).

In some embodiments, the method comprises: (a) administering the5-azacytidine daily to the human for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days; (b) administering the atleast one additional therapeutic agent to the human for one or moredays; and (c) optionally repeating steps (a) and (b).

In some embodiments, the method comprises: (a) administering the5-azacytidine daily to the human for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, or 14 days of a 28-day cycle; (b) concurrently administering theat least one therapeutic agent daily to the human for 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, or 28 days of a 28-day cycle; and (c) optionally repeating steps(a) and (b). In some embodiments, wherein administering the at least oneadditional therapeutic agent comprises administering gilteritinib,midostaurin, quizartinib, enasidenib, ivosidenib, or venetoclax.

In some embodiments, the method comprises: (a) administering the5-azacytidine daily to the human for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days of a 28-day cycle; (b)concurrently administering the at least one additional therapeutic agentdaily to the human for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days of a28-day cycle; and (c) optionally repeating steps (a) and (b). In someembodiments, wherein administering the at least one additionaltherapeutic agent comprises administering gilteritinib, midostaurin,quizartinib, enasidenib, ivosidenib, or venetoclax.

In some embodiments, the method comprises the sequential steps of: (a)administering the 5-azacytidine to the human for 7 days of a 28-daycycle; (b) administering the at least one additional therapeutic agentto the human for 1 day of a 28-day cycle; (c) administering5-azacytidine to the human for 6 days of a 28-day cycle; and (d)repeating steps (a) to (c) after 7 days of a resting period.

In some embodiments, the method comprises the sequential steps of: (a)administering 5-azacytidine daily to the human for 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days of a 28-daycycle; (b) administering the at least one additional therapeutic agentdaily to the human for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, or 21 days of a 28-day cycle; (c) administering5-azacytidine daily to the human for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days of a 28-day cycle; and(d) optionally repeating steps (a) and (c) after 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days of a restingperiod.

In some embodiments, the human has acute myeloid leukemia. In someembodiments, the human has myelodysplastic syndrome. In someembodiments, the myelodysplastic syndrome is high and very high riskmyelodysplastic syndromes as defined by the Revised InternationalPrognostic Scoring System (IPSS-R).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents the maximum EC₅₀ fold shift of 5-azacytidine incombination with gilteritinib, and 5-azacytidine in combination withmidostaurin; both with cell lines MV4-11 and MOLM-13. The results fromthree different dosing schedules are shown: (i) 5-azacytidineadministered first (black bar); (ii) two agents administeredconcurrently (light gray bar); and (iii) 5-azacytidine administeredsecond (medium gray bar).

FIG. 2 represents the three different dosing schedules of (i)5-azacytidine (AZA) administered first at intervals before the FLT3inhibitor (FLT3i); (ii) the two agents (5-azacytidine and FLT3i)administered concurrently; and (iii) 5-azacytidine administered secondat intervals after the FLT3i is administered. The FLT3 inhibitor may beany suitable FLT3 inhibitor, including midostaurin, or gilteritinib.

FIGS. 3A-D represent the maximum EC₅₀ fold shift of 5-azacytidine incombination with venetoclax with cell lines MV4-11 (FIG. 3A) and MOLM-13(FIG. 3C). The results from three different dosing schedules are shown,as indicated in the legend: (i) 5-azacytidine administered first (blackbar); (ii) the two agents administered concurrently (light gray bar);and (iii) 5-azacytidine administered second (medium gray bar). A synergyindex is also shown for 5-azacytidine administered in combination withvenetoclax with cell lines MV4-11 (FIG. 3B) and MOLM-13 (FIG. 3D) forthe three different dosing schedules.

FIGS. 4A-C represent Response Surface Analyses showing synergisticeffects of 5-azacytidine with venetoclax in MV4-11 cells when5-azacytidine is administered first (FIG. 4A), the relatively lowersynergy with simultaneous administration (FIG. 4B), and synergy withvenetoclax administered first (FIG. 4C). Response surface methodology(RSM) is a well-known statistical method to explore the relationshipsbetween several explanatory variables and one or more responsevariables. RSM uses a sequence of designed experiments to obtain anoptimal response, which in the present case is the synergistic effectsof 5-azacytidine with venetoclax.

FIG. 5 depicts a western blot showing that (a) 5-azacytidine andmidostaurin (“aza+0.3 μM Mido”) and (b) 5-azacytidine and gilteritinib(“aza+0.3 μM Gilt”) augment MCL-1 degradation in MV4-11 cell lines.

FIG. 6 depicts a western blot showing that 5-azacytidine and venetoclaxtreatment decreases MCL-1 levels in FLT3ITD MV4-11 cells.

FIGS. 7A-C depicts in vivo assessments of 5-azacytidine combinations ina MOLM-13 xenograft model, with a graph of percent survival (y-axis) vs.day 0 to 70 (x-axis). FIG. 7A shows the results of the combination of5-azacytidine and midostaurin, FIG. 7B shows the results of5-azacytidine combined with venetoclax, and FIG. 7C shows the results ofthe combination of 5-azacytidine and gilteritinib. Dosing for theexperiments shown in FIGS. 7A-C was as follows: (i) 5-azacytidine (lowexposure, extended duration, LEED): 1 mg/kg interperitoneally (IP), oncedaily for five days, three times (qdx 5×3); (ii) 5-azacytidine (highexposure, limited duration, HELD): 3 mg/kg interperitoneally (IP), oncedaily for five days (qd×5); (iii) Midaustaurin (100 mg/kg orally (PO),once daily for twenty-one days (qd×21)); (iv) Gilteritinib (4 mg/kgorally (PO), once daily for twenty-one days (qd×21)); and (v) Venetoclax(100 mg/kg orally (PO), once daily for twenty-one days (qd×21)). P-value(relative to best single agent) *P<0.05; **P<0.001; ***P<0.0001.

FIGS. 8A, 8B, and 8C depict the sensitivity of 22 AML cell lines to5-azacytidine (AZA) and venetoclax as single agents (FIGS. 8A and 8B,respectively) and the combination with 5-azacytidine and venetoclax(FIG. 8C).

FIGS. 9A-F show the cell survival of MV4-11 cells (FIGS. 9A-C) andMOLM-13 cells (FIGS. 9D-F) and the corresponding synergy index (SI)seven days after the start of treatment with 5-azacytidine andvenetoclax. The following schedules were tested: 5-azacytidineadministration on Days 1, 2 and 3, followed by venetoclax administrationon Day 4 (5-azacytidine (AZA) First) (FIGS. 9A and 9D); 5-azacytidineand venetoclax co-administration on Day 1, followed by 5-azacytidineadministration on Days 2 and 3 (Simultaneous) (FIGS. 9B and 9E); andvenetoclax administration on Day 1, followed by 5-azacytidine on Days 2,3 and 4 (venetoclax first) (FIGS. 9C and 9F).

FIGS. 10A, 10B, and 10C depict the correlation of MCL-1 expression withthe degree of the synergistic effect of the combination of 5-azacytidineandvenetoclax in a panel of engineered BaF3 cell lines expressingwild-type FLT3, FLT3-ITD or FLT3 (D835Y) mutations.

FIG. 11 depicts the correlation of MCL1 RNA level, as measured byRNASeq, with the synergy index (r²=−0.5607, p=0.0101) in a panel of 20AML cell lines.

FIG. 12A-H depict the extent of 5-azacytidine-mediated MCL-1 degradationin four different AML cell lines: KG1α (FIG. 12A), MV4-11 (FIG. 12B),THP-1 (FIG. 12C), and OCI-AML2 (FIG. 12D). The results showed5-azacytidine-venetoclax synergistic activity with KG1α (FIG. 12E) andMV4-11 (FIG. 12F) cell lines (synergy index (SI) of 70 and 35.5,respectively) and very little or no synergistic activity with THP-1(FIG. 12G) and OCI-AML-2 (FIG. 12H) cell lines (SI of 20.2 and 10.8,respectively). For the KG1α (FIG. 12A) and MV4-11 (FIG. 12B) cell lines,where 5-azacytidine-venetoclax had the greatest synergistic effect(FIGS. 12E and 12F), 5-azacytidine led to MCL-1 degradation the fastest,starting 6 hours after treatment. In contrast, for THP-1 (FIG. 12G),where 5-azacytidine-venetoclax only provided minor synergistic activityshowed 5-azacytidine-mediated MCL-1 degradation later, starting at 16hours, with incomplete degradation by 24 hours (FIG. 12C). For OCI-AML2(FIG. 12D), where 5-azacytidine-venetoclax had the lowest synergisticeffect (FIG. 12H), 5-azacytidine treatment did not lead to anydegradation of MCL-1.

FIG. 13A depicts a western blot evaluating the degradation of caspase 3.Cells were treated with Z-VAD-FMK, a pan-caspase inhibitor, and theextent of MCL-1 degradation by 5-azacytidine was measured. FIG. 13Bshows a bar graph of MCL-1 degradation by 5-azacytidine, where cellswere treated with 20 μM Z-VAD-FMK for 1 hour before 5-azacytidinetreatment for another 16 hours. Caspase inhibition partially ablatedMCL-1 degradation by 5-azacytidine in MV4-11 cells, which suggestedadditional, caspase-independent mechanisms of MCL-1 degradation.

FIGS. 14A and B depict the results of an RNAseq performed on MV4-11cells treated with PBS (vehicle), 1 μM 5-azacytidine for 24 hours (FIG.14A), or with 1 μM 5-azacytidine for 48 hours (FIG. 14B). FIGS. 14A andB show volcano plots of significantly modified genes at 24 hours (FIG.14A) and 48 hours (FIG. 14B), showing that 5-azacytidine induced 133differentially expressed genes at 24 hours and 226 differentiallyexpressed genes at 48 hours. Upon further analysis of the5-azacytidine-induced differentially expressed genes, two genes wereidentified that have previously been shown to regulate MCL1 expression:activating transcription factor 3 (ATF3) and stearoyl-CoA desaturase(SCD). ATF3 expression was increased two-fold 48 hours after5-azacytidine treatment. The expression of SCD (Stearoyl-CoAdesaturase), a regulator of lipid metabolism and MCL1, was decreased2.5-fold by 5-azacytidine treatment at 48 hours. Alterations in ATF3(FIG. 14C) and SCD (FIG. 14D) expression were validated in a separateexperiment using real-time PCR. ATF3 expression was increased in a time-and concentration-dependent manner, as 0.3 μM 5-azacytidine treatmentwas not sufficient to induce ATF3 expression at either 24 or 48 hours(FIG. 14C). Similarly, SCD expression was decreased rapidly within 24hours when treated with 3 μM 5-azacytidine, although it was not affectedby low concentrations of 5-azacytidine at this timepoint (FIG. 14D).

FIGS. 15A-C shows the results of siRNA knockdown of ATF3 and/or SCDgenes in MV4-11 cells to assess their function in synergy. MV4-11 cellswere left untransfected or transfected with ATF3, SCD, or control(scrambled) siRNA. As a control, cells were transfected with siRNA andcollected for RNA and qPCR 72 hours after transfection (FIG. 15A). Incells treated with scramble siRNA, no changes in ATF3 (FIG. 15B) or SCD(FIG. 15C) expression were seen. Following transfection, cells weretreated with various concentrations of 5-azacytidine daily for Days 1-3.At Day 4, cells were dosed with venetoclax, followed by cell viabilitytest using CellTiter-Glo® 7 after treatment initiation.5-azacytidine-venetoclax synergy was calculated using Combenefit andHighest Single Agent analysis (FIG. 15 D-G). FIG. 15D=untransfectedcells; FIG. 15E=scrambled RNAi; FIG. 15F=ATF3 knockdown; and FIG.15G=SCD knockdown.

FIGS. 16A, 16B, 16C, 16D, 16E, and 16F depict the results of anevaluation as to whether 5-azacytidine and venetoclax have synergisticactivity in vivo at doses and schedules corresponding to injectable5-azacytidine (HELD) or oral 5-azacytidine (LEED). MV4-11 (FIGS. 16A-C)and MOLM-13 (FIGS. 16D-F), two cell lines that showed5-azacytidine-venetoclax synergy, were to used to generate disseminatedAML xenograft mice in immunodeficient animals. In vitro, venetoclaxsensitized both cell lines to venetoclax (FIGS. 16A and 16D) andsynergized with 5-azacytidine (FIGS. 16B and 16E). To model oral5-azacytidine (LEED) regimens, mice were treated with 1 mg/kg5-azacytidine for 15 days (low exposure, extended duration).Alternatively, to use the same cumulative dose but with an injectable5-azacytidine (HELD) regimen, mice were treated with 3 mg/ml5-azacytidine for 5 days (high exposure, limited duration).

FIGS. 17A, 17B, 17C, 17D, 17E, 17F, 17G, 17H, 17I, 17J, and 17K depictthe results of an investigation as to whether co-treatment with5-azacytidine and FLT3 inhibitors have a synergistic effect in AMLcells. FIGS. 17A-D show the results from experiments with MV4-11 cells.FIGS. 17E-H show the results from experiments with MOLM-13 cells. FIGS.17A, 17B, 17E, and 17F show the results from treatment with5-azacytidine and midostaurin. FIGS. 17C, 17D, 17G, and 17H show theresults from treatment with 5-azacytidine and gilteritinib. Cells weretreated with daily doses of 5-azacytidine on Day 1-3, and then treatedwith a FLT-3 inhibitor (midostaurin or gilteritinib) at Day 4. Cellswere collected on Day 7 and cell viability was assessed byCellTiter-Glo® assay. Midostaurin sensitized MV4-11 to 5-azacytidine(FIG. 17A) and showed synergistic activity with 5-azacytidine (FIG.17B). Similar effects were observed in MV4-11 cells treated with5-azacytidine and gilteritinib (FIGS. 17C and 17D), as well as inMOLM-13 cells treated with 5-azacytidine and midostaurin (FIGS. 17E and17F) or gilteritinib (FIGS. 17G and 17H). FIG. 17I shows the results inMOLM-13 cells of percent survival (y-axis) vs day 1-70 foradministration of vehicle, 5-azacytidine (LEED), 5-azacytidine (HELD),midostaurin, 5-azacytidine (LEED) and midostaurin, and 5-azacytidine(HELD) and midostaurin. FIG. 17J shows the results in MV4-11 cells ofpercent survival (y-axis) versus day 1-91 for administration of vehicle,5-azacytidine (LEED), 5-azacytidine (HELD), midostaurin, 5-azacytidine(LEED) and midostaurin, and 5-azacytidine (HELD) and midostaurin. FIG.17K shows the results in MOLM-13 cells of percent survival (y-axis)versus day 1-70 for administration of vehicle, 5-azacytidine (LEED),5-azacytidine (HELD), gilteritinib, 5-azacytidine (LEED) andgilteritinib, and 5-azacytidine (HELD) and gilteritinib.

FIG. 18 depict the results the overall study design of Example 3.

DETAILED DESCRIPTION I. Overview

The present disclosure is based in part on the surprising discovery that5-azacytidine primes cancer cell lines for apoptosis in combination withother agents to treat cancer. In particular, 5-azacytidine was found toreduce levels of myeloid cell leukemia 1 (MCL-1), resulting in primingcancer cells to be responsive to FMS-like tyrosine kinase 3 (FLT3) orvenetoclax activity. Low baseline levels of MCL-1 correlate with bettersynergistic responses to treating cancer, as it may be easier to reduceMCL-1 levels below a certain threshold when baseline levels are alreadyreduced. Thus, it was surprisingly found that 5-azacytidine incombination with other therapeutic agents, such as FLT3 inhibitors orvenetoclax, reduce MCL-1 levels more than each agent alone.

Another aspect of the methods disclosed herein is that the 5-azacytidineis administered orally in combination with other agents that are alsoadministered orally. Thus, the 5-azacytidine oral dosing protocol incombination with other oral agents of the present disclosure produces abetter patient experience as well as reduced risk of hospitalinfections, which can be particularly risky for cancer patients withcompromised immune systems.

Yet another aspect of the methods disclosed herein is that the5-azacytidine is given orally at a relatively low dose for a prolongedperiod of time (e.g. 1 mg/kg, once daily for fifteen days (QD×15) and 1mg/kg, once daily for five days, three times (qd×5×3)) in combinationwith one or more agents. Prior to the present disclosure, the rationalefor administering 5-azacytidine by injection at relatively high doses(e.g., 75 mg/m² subcutaneously or intravenously, once daily for sevendays every four weeks) was because it was thought that high doses ofchemotherapy agents, such as 5-azacytidine, would result in greatertumor cell death and therefore longer patient survival. The presentdisclosure is contrary to this “conventional wisdom.” In particular, itwas surprisingly found that relatively low oral doses of 5-azacytidineover a prolonged dosing period in combination with one or more agentsresult in therapeutic efficacy in treating AML and/or MDS. Althoughlower doses of 5-azacyitidine are administered in combination with othertherapeutic agents, the combination therapy surprisingly providestherapeutic efficacy in treating AML and/or MDS.

Regarding cell differentiation, without being bound by theory, it ishypothesized that a decrease in 5-azacytidine dose may result indemethylation of cancer cells. Chemotherapy drugs triggeringdifferentiation are thought to turn blast cells from the bone marrowinto circulation and make them into functional cells and reduce theirlife span. Further, it is thought that exposure to 5-azacytidine wouldturn blast cells into terminally differentiated immune cells, resultingin killing tumor cells (hematopoietic differentiation pathway); once theblast cells are pushed down the differentiation lineage, they ultimatelykill tumor cells. In sum, it is thought that the lower dose of5-azacytidine given over a longer period of time with one or more othertherapeutic agents, such as FLT3 inhibitors or venetoclax, results ingreater differentiation and ultimately more significant tumor celldeath.

Further, as detailed in the examples below, it was surprising found thatin some instances, AML cell lines that were resistant to venetoclaxactivity were found to be responsive to ventoclax after treatment withthe combination of 5-azacytidine and venetoclax, wherein the5-azacytidine is administered first. Also, the combination of5-azacytidine and a FLT3 inhibitor or venetoclax provided a synergisticeffect and reduced MCL-1 protein levels more than each agent alone. Itwas also found that levels of MCL-1 expression correlated with thesynergy of the 5-azacytidine-venetoclax combination (e.g., the lower thelevels of MCL-1 expression, the greater the synergistic effect).

In one aspect, the present disclosure is directed to methods of treatinga human having acute myeloid leukemia (AML) by administering to thehuman (i) a pharmaceutical composition comprising 5-azacytidineadministered orally; and (ii) at least one additional therapeutic agent.In some embodiments, the additional therapeutic agent comprisesgilteritinib, midostaurin, quizartinib, enasidenib, ivosidenib, and/orvenetoclax. Also disclosed herein are (i) pharmaceutical compositionscomprising 5-azacytidine administered orally, and (ii) at least oneadditional therapeutic agent for treating AML in a human.

In some embodiments, certain combinations work synergistically in thetreatment of particular diseases or disorders, including, e.g., types ofcancer and certain diseases and conditions associated with, orcharacterized by, undesired angiogenesis or abnormal cell proliferation.

Acute myeloid leukemia (AML), also known as acute myelogenous leukemia,is an aggressive, heterogeneous, myeloid malignancy. According to theAmerican Cancer Society, AML is the most common type of leukemiadiagnosed in adults and makes up 32% of all adult leukemia cases. It isestimated that approximately 19,940 people will be diagnosed with AML in2020 in the United States (US) with 11,180 patients estimated to diefrom the disease. The disease is particularly difficult to treat inolder adults who account for the majority of patients; thus, thefive-year overall survival is only approximately 29%. National CancerInstitute, SEER Cancer Stat Facts: Leukemia—Acute Myeloid Leukemia(AML), https://seer.cancer.gov/statfacts/html/amyl.html (accessed 10Jun. 2020). Since the 1970s, initial standard therapy, for those fitenough to receive it, consisted of the ‘7+3’ regimen, which includesseven days of continuous infusion cytarabine and three days of ananthracycline. Rai et. al. Blood 1981:58: 1203-1212. Over the next 35years, a number of clinical trials attempting to augment the safety andefficacy of AML treatment have been performed with little change in thestandard of care. However, recent data detailing the molecular ontogenyof AML have elucidated causal pathways which have led to efforts todevelop targeted drug therapies. E. Winer and R. Stone, Ther. Adv.Hematol., 10:PMC6624910 (July 2019).

There is a long felt need for the combination treatments describedherein, as AML has a high rate of relapse. Additionally, relapsed andrefractory AML is a disease that is very difficult to treat and islikely driven by multiple abnormal signaling pathways that give theleukemic cell an advantage in overcoming any single pathway that isbeing inhibited. Thus, successful combination treatments are highlydesirable to treat AML patients.

In one aspect of the methods of treatment described herein, the patientto be treated is about age 60 or older. In another aspect of the methodsof treatment described herein, the patient to be treated is about age 65or older, about age 70 or older, about age 75 or older, or about age 80or older. In yet another aspect, the patient is a relapsed AML patient.In another aspect, the patient is a refractory AML patient. The patientto be treated can also be under about age 60, under about age 55, underabout age 50, under about age 45, or under about age 40. In otheraspects, the patient to be treated has FLT3 mutations, either FLT3-ITDor FLT3-TKD. In some aspects, the patient to be treated has a recurrentAML mutation. Exemplary AML mutations include, but are not limited to,FMS-related tyrosine kinase 3 (FLT3), Kirsten rat sarcoma viral oncogenehomolog (KRAS), neuroblastoma RAS viral (V-Ras) oncogene homolog (NRAS),proto-oncogene c-Kit (KIT), protein tyrosine phosphatase non-receptortype 11 (PTPN11), neurofibromin 1 (NF1), DNA methyltransferase 3A(DNMT3A), isocitrate dehydrogenase 1 (IDH1), isocitrate dehydrogenase 2(IDH2), ten-eleven translocation-2 (TET2), additional sex comb-like 1(ASXL1), enhancer of zeste homolog 2 (EZH2), mixed-lineage leukemia1/histone-lysine N-methyltransferase 2A (MLL/KMT2A), nucleophosmin(NPM1), CCAAT enhancer binding protein alpha (CEBPA), runt-relatedtranscription factor 1 (RUNX1), GATA-binding factor 2 (GATA2), tumorprotein p53 (TP53), serine and arginine rich splicing factor 2 (SRSF2),U2 small nuclear RNA auxiliary factor 1 (U2AF1), splicing factor 3bsubunit 1 (SF3B1), zinc finger (CCCH type), RNA-binding motif andserine/arginine rich 2 (ZRSR2), RAD21 cohesin complex component (RAD21),stromal antigen 1 (STAG1), stromal antigen 2 (STAG2), structuralmaintenance of chromosomes 1A (SMC1A), and structural maintenance ofchromosomes protein 3 (SMC3).

In another aspect, the present disclosure is directed to methods oftreating a human having myelodysplastic syndrome (MDS) by administeringto the human (i) a pharmaceutical composition comprising 5-azacytidineadministered orally; and (ii) at least one additional therapeutic agent.In some embodiments, the additional therapeutic agent comprisesgilteritinib and/or venetoclax. Also disclosed herein are (i)pharmaceutical compositions comprising 5-azacytidine administeredorally, and (ii) at least one additional therapeutic agent for treatingMDS in a human.

In some embodiments, the MDS is a higher risk or high risk MDS.Higher-risk MDS for this disclosure is defined as High or Very High riskaccording to the Revised International Scoring System (IPSS-R) (Voso M.T., et. al., J Clin Oncol. 2013; 31(21): 2671-2677; and Greenberg P. L.et al., Blood. 2012, 120(12): 2454-2465), where these patients havemedian survival of 1.6 and 0.8 years respectively.

A. Overview of Experimental and Clinical Study Protocols

Example 2 details a clinical study analyzing the relationships of drugexposure with efficacy, safety, pharmacodynamics, includingestablishment of a maximum tolerated dose (MTD) or a maximumadministered dose (MAD) in AML, patients. The minimal residual diseasein blood and/or bone marrow following treatment will also be determined.The study population consists of AML patients who are in first relapse,refractory to 1 or 2 standard induction treatments, or newly diagnosedAML patients who are not candidates to receive intensive IC. The drugcombinations to be tested include oral 5-azacytidine when given incombination with ivosidenib, enasidenib, venetoclax, or an FLT3inhibitor, such as gilteritinib (in AML patients with a FLT3 ITD or TKDmutation).

Isocitrate dehydrogenase (IDH) is a critical enzyme in the citric acidcycle. Mutated forms of IDH produce high levels of the (R)-enantiomer of2-hydroxyglutarate (R-2-HG) and can contribute to the growth of tumors.IDH1 catalyzes this reaction in the cytoplasm, while IDH2 catalyzes thisreaction in mitochondria. Ivosidenib and enasidenib are IDH inhibitors.

Ivosidenib (Tibsovo®) is a small molecule inhibitor of IDH1. In tumorsfrom patients diagnosed with glioma, Acute Myeloid Leukemia (AML),cholangiocarcinoma, and chondrosarcoma, somatic mutations in theconserved active site of isocitrate dehydrogenase (IDH) 1 and 2 areobserved. With these new mutations, these enzymes exhibit new,neomorphic behavior, which results in the reduction of α-ketoglutarateto the oncometabolite R-2-hydroxyglutarate. Ivosidenib competitivelyinhibits α-ketoglutarate-dependent enzymes, ultimately leading toepigenetic alterations and impaired hematopoietic differentiation.

In in vitro studies, ivosidenib showed non-competitive inhibitorybehavior towards the alpha-ketoglutarate (α-KG) substrate and to theNADPH cofactor. This is what is believed to lead to ivonsidenib being arapid equilibrium inhibitor of the mutated isocitrate dehydrogenase 1(mIDH1)-R132H homodimer.

Enasidenib (Idhifa®) is a small molecule inhibitor of the isocitratedehydrogenase 2 (IDH2) gene. As noted above, mutated forms of IDHproduce high levels of R-2-HG, with IDH1 catalyzing this reaction in thecytoplasm and IDH2 catalyzing this reaction in mitochondria. Enasidenibdisrupts this cycle by decreasing total (R)-2-HG levels in themitochondria

Venetoclax (Venclexta® and Venclyxto®) is a BH3 (Bcl-2 homology domain3)-mimetic as it blocks the anti-apoptotic B-cell lymphoma-2 (Bcl-2)protein, leading to programmed cell death of chronic lymphocyticleukemia (CLL) cells. Overexpression of Bcl-2 in some lymphoidmalignancies has sometimes shown to be linked with increased resistanceto chemotherapy.

FLT3 inhibitors are tyrosine kinase inhibitors (TKI). Like othertyrosine kinase inhibitors, FLT3 inhibitors compete for the adenosinetriphosphate (ATP) binding site in the active domain of the kinase,which inhibits the ability of the protein to be phosphorylated, andsubsequently decreases the activity of that protein. FLT3 mutations areone of the most common findings in patients with acute myeloid leukemia(AML). The FLT3/ITD gene is found in approximately 30% of patients withAML with normal cytogenetics. The FLT3 gene is expressed mainly in humanhematopoietic progenitors and dendritic cells and plays key roles inleukemia cell proliferation, differentiation, and survival. Constitutiveactivation of the FLT3/ITD gene triggers multiple downstream signalingcascades, such as STATS, RAS, MEK, and PI3K/AKT pathways, and ultimatelycauses suppression of apoptosis and differentiation of leukemic cells,including dysregulation of leukemic cell proliferation. The FLT3inhibitors evaluated include midostaurin (Rydapt®) andgilteritinib)(Xospata®. Midostaurin is a semi-synthetic derivative ofstaurosporine, an alkaloid from the bacterium Streptomycesstaurosporeus, and is active against oncogenic CD135 (FMS-like tyrosinekinase 3 receptor, FLT3). Gilteritinib acts as an inhibitor of AXLreceptor tyrosine kinase.

Example 3 details a Phase 1b/2 clinical study evaluating the safety,tolerability, and preliminary efficacy of oral 5-azacytidine incombination with biomarker directed therapies in patients with acutemyeloid leukemia (AML) or higher-risk myelodysplastic syndromes(HR-MDS). The study population consists of patients with newly diagnosedAML not eligible for intensive induction chemotherapy andrelapsed/refractory (R/R) AM and treatment of subjects with primary orsecondary MDS who are Revised International Prognostic Scoring System(IPSS-R) high and very high risk. The drug combinations to be testedinclude oral 5-azacytidine when given in combination with venetoclax orgilteritinib.

B. 5-Azacytidine

5-Azacytidine (National Service Center designation NSC-102816; CASRegistry Number 320-67-2) is also known as azacitidine, abbreviated asAZA, or 4-amino−1-B-D-ribofuranosyl-1,3,5-triazin-2(1H)-one. Themarketed product VIDAZA® (5-azacytidine for injection) contains5-azacytidine, and is for subcutaneous or intravenous use. 5-Azacytidineis a pyrimidine nucleoside analog of cytidine. 5-Azacytidine has thefollowing structure:

After its incorporation into replicating DNA, 5-azacytidine forms acovalent complex with DNA methyltransferases. DNA methyltransferases areresponsible for de novo DNA methylation and for reproducing establishedmethylation patterns in daughter DNA strands of replicating DNA.Inhibition of DNA methyltransferases by 5-azacytidine leads to DNAhypomethylation, thereby restoring normal functions to morphologicallydysplastic, immature hematopoietic cells and cancer cells byre-expression of genes involved in normal cell cycle regulation,differentiation and death. The cytotoxic effects of these cytidineanalogs cause the death of rapidly dividing cells, including cancercells, that are no longer responsive to normal cell growth controlmechanisms. 5-azacytidine also incorporates into RNA. The cytotoxiceffects of 5-azacytidine may result from multiple mechanisms, includinginhibition of DNA, RNA and protein synthesis, incorporation into RNA andDNA, and activation of DNA damage pathways.

Injectable 5-azacytidine has been tested in clinical trials and showedsignificant anti-tumor activity, such as, for example, in the treatmentof myelodysplastic syndromes (MDS), acute myelogenous leukemia (AML),chronic myelogenous leukemia (CML), acute lymphocytic leukemia (ALL),and non Hodgkin's lymphoma (NHL). See, e.g., Aparicio et al., Curr.Opin. Invest. Drugs 3(4): 627-33 (2002).

5-Azacytidine is approved for subcutaneous (SC) or intravenous (IV)administration to treat patients with the followingFrench-American-British (FAB) myelodysplastic syndrome subtypes:refractory anemia (RA) or refractory anemia with ringed sideroblasts (ifaccompanied by neutropenia or thrombocytopenia or requiringtransfusions), refractory anemia with excess blasts (RAEB), refractoryanemia with excess blasts in transformation (RAEB-T), and chronicmyelomonocytic leukemia (CMMoL). Oral dosing has been studied inclinical trials, such as NCT00761722, NCT01519011, NCT00528982, andNCT01757535. Oral formulations and methods of treatment using5-azacytidine are disclosed in U.S. Pat. No. 8,846,628, which isincorporated by reference in its entirety.

In some embodiments, 5-azacytidine is administered orally. In someembodiments, 5-azacytidine is administered in the form of a capsule or atablet. In some embodiments, the tablet is a non-enteric-coated tablet.In some embodiments, the 5-azacytidine is administered at a dose ofabout 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, about250 mg, about 260 mg, about 270 mg, about 280 mg, about 290 mg, about300 mg, about 310 mg, about 320 mg, about 330 mg, about 340 mg, about350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, about400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg, about450 mg, about 460 mg, about 470 mg, about 480 mg, about 490 mg, about500 mg, about 510 mg, about 520 mg, about 530 mg, about 540 mg, about550 mg, about 560 mg, about 570 mg, about 580 mg or about 600 mg orally.In some embodiments, the 5-azacytidine is administered at a dose ofabout 200 to about 300 mg orally. In some embodiments, the 5-azacytidineis administered at a dose of about 200 mg. In some embodiments, the5-azacytidine is administered at a dose of about 300 mg. In someembodiments, the 5-azacytidine is administered at a dose of 50 mg, 60mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg,160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg,250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, 310 mg, 320 mg, 330 mg,340 mg, 350 mg, 360 mg, 370 mg, 380 mg, 390 mg, 400 mg, 410 mg, 420 mg,430 mg, 440 mg, 450 mg, 460 mg, 470 mg, 480 mg, 490 mg, 500 mg, 510 mg,520 mg, 530 mg, 540 mg, 550 mg, 560 mg, 570 mg, 580 mg or 600 mg orally.In some embodiments, the 5-azacytidine is administered at a dose of 200to 300 mg orally. In some embodiments, the 5-azacytidine is administeredat a dose of 200 mg. In some embodiments, the 5-azacytidine isadministered at a dose of 300 mg. In some embodiments, 5-azacytidine isadministered daily orally for the first seven, fourteen, or twenty-onedays of a 28-day cycle. In some embodiments, 5-azacytidine isadministered daily orally for the first fourteen days of a 28-day cycle.In some embodiments, 5-azacytidine administered to the subject once perday. In some embodiments, 5-azacytidine administered to the subject twotimes per day.

In some embodiments, the 5-azacytidine is administered orally at a doseof about 200 mg per day for 14 days in a 28-day cycle. In someembodiments, the 5-azacytidine is administered orally at a dose of 200mg per day for 14 days in a 28-day cycle. In some embodiments, the5-azacytidine is administered orally at a dose of about 300 mg per dayfor 14 days in a 28-day cycle. In some embodiments, the 5-azacytidine isadministered orally at a dose of 300 mg per day for 14 days in a 28-daycycle. In some embodiments, the 5-azacytidine is administered orally ata dose of about 200 mg per day for 21 days in a 28-day cycle. In someembodiments, the 5-azacytidine is administered orally at a dose of 200mg per day for 21 days in a 28-day cycle. In some embodiments, the5-azacytidine is administered orally at a dose of about 300 mg per dayfor 21 days in a 28-day cycle. In some embodiments, the 5-azacytidine isadministered orally at a dose of 300 mg per day for 21 days in a 28-daycycle.

In some embodiments, the 5-azacytidine is administered orally daily for1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or greater than 14 days,optionally followed by a treatment dosing holiday of 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, or greater than 14 days. In someembodiments, the 5-azacytidine is administered orally daily for 14 ormore days, optionally followed by a treatment dosing holiday of 7 ormore days. In some embodiments, the 5-azacytidine is administered orallyfor 21 or more days, optionally followed by a treatment dosing holidayof 7 or more days. In some embodiments, the 5-azacytidine isadministered orally for 14 days, optionally followed by a treatmentdosing holiday of 14 days. In some embodiments, the 5-azacytidine isadministered orally for 21 or more days, followed by a treatment dosingholiday of 7 or more days. In some embodiments, the 5-azacytidine isadministered orally for 14 days, followed by a treatment dosing holidayof 14 days.

In some embodiments, the 5-azacytidine is administered orally at a doseof about 300 mg daily for 14 days, followed by a treatment dosingholiday of 14 days. In some embodiments, the 5-azacytidine isadministered orally at a dose of 300 mg daily for 14 days, followed by atreatment dosing holiday of 14 days. In some embodiments, the5-azacytidine is administered orally at a dose of about 200 mg daily for14 days, followed by a treatment dosing holiday of 14 days. In someembodiments, the 5-azacytidine is administered orally at a dose of 200mg daily for 14 days, followed by a treatment dosing holiday of 14 days.In some embodiments, the 5-azacytidine is administered orally at a doseof about 300 mg daily for 21 days, followed by a treatment dosingholiday of 7 days. In some embodiments, the 5-azacytidine isadministered orally at a dose of 300 mg daily for 21 days, followed by atreatment dosing holiday of 7 days. In some embodiments, the5-azacytidine is administered orally at a dose of about 200 mg daily,followed by a treatment dosing holiday of 7 days. In some embodiments,the 5-azacytidine is administered orally at a dose of 200 mg daily,followed by a treatment dosing holiday of 7 days.

In some embodiments, the 5-azacytidine is administered orally using atreatment cycle comprising administration of 5-azacytidine per day for 7days in a 28-day cycle. In some embodiments, the 5-azacytidine isadministered orally using a treatment cycle comprising administration of5-azacytidine per day for 14 days in a 28-day cycle. In someembodiments, the 5-azacytidine is administered orally using a treatmentcycle comprising administration of 5-azacytidine per day for 21 days ina 28-day cycle.

5-azacytidine exerts effects on cell viability and epigeneticreprogramming of cells. Taylor and Jones, Cell 20(1):85-93 (1980). Athigh doses, 5-azacytidine is thought to exercise a predominantly acutecytotoxic effect (Khan et al., Experimental Hematology 36(2): 149-57,2008), while at low doses it inhibits clonogenicity of tumor cellsthough differentiation (Tsai et al., Cancer Cell, 21(3): 430-46, 2012).

The marketed product VIDAZA®, the injectable formulation of5-azacytidine, is administered at relatively higher doses and forshorter duration compared to the oral, non-enteric coated formulation of5-azacytidine as described in U.S. Pat. No. 8,846,628, including CC-486.Clinical studies revealed that CC-486 induces more sustainabledemethylative effects as compared to VIDAZA® (Laille et al., PLOSOne10(8):e0135520, 2015), although cumulative exposures of 14 or 21 dayregimens of CC-486 are lower than VIDAZA®, administered for 5 days(Garcia-Manero et al., Leukemia 30(4):889-96, 2016).

To examine differences in cytotoxic and epigenetic effects as a functionof duration of exposure to 5-azacytidine, high exposure, limitedduration (HELD) administration of injectable 5-azacytidine was comparedwith low exposure, extended duration (LEED) administration of5-azacytidine administered orally. To model injectable and oralazacytidine dosing in non-clinical systems, the total 5-azacytidineexposure was held constant while varying the duration of exposure. Insome embodiments, to model the oral administration of 5-azacytidine, the5-azacytidine was delivered at a low exposure for extended duration(LEED), at a dose of 1 mg/kg, once daily for fifteen days (QD×15). Tomodel the same cumulative dose by intravenous or subcutaneousadministration of 5-azacytidine, the 5-azacytidine was administered at ahigh exposure for a limited duration (HELD), at a dose of 3 mg/kg, oncedaily for five days (QD×5).

In some embodiments, LEED administration of 5-azacytidine in combinationwith other agents provides a sustained pharmacodynamic effect and/orimproved patient compliance. A sustained pharmacodynamic effect mayinclude any change elicited by 5-azacytidine, which includes for exampleMCL-1 degradation, and/or changes in ATF3 or SCD gene expression. Insome embodiments, LEED of 5-azacyitidine in combination with otheragents provides a reduction in global DNA methylation (e.g., due toincreased nucleic acid incorporation) that sustained through the end ofthe treatment cycle (i.e., a 28-day cycle) compared to HELD of5-azacyitidine in combination with other agents. In some embodiments,LEED of 5-azacyitidine in combination with other agents provides adifferentiation maker upregulation that peaks at Day 21 of a 28-daycycle and has a cell death that is characterized by a gradual loss ofviability through Day 28 of a 28-day cycle. In some embodiments, HELD of5-azacyitidine in combination with other agents provides adifferentiation marker upregulation that peaks at Day 7 of a 28-daycycle and has a cell death that is characterized by a peak at Day 14followed by recovery in a 28-day cycle. In some embodiments, LEED of5-azacyitidine in combination with other agents provides a higherexpression of myeloid differentiation markers, which include but are notlimited to CD11b, CD14, CD86, HLA-DR and MERTK, that is sustainedthrough a treatment cycle (i.e., a 28-day cycle) compared to HELD of5-azacyitidine in combination with other agents. In some embodiments,LEED of 5-azacyitidine in combination with other agents provides morepronounced epigenetic changes and more extensive differentiationcompared to HELD of 5-azacyitidine in combination with other agents.

II. Pharmaceutical Formulations A. Overview

Embodiments herein encompass pharmaceutical formulations andcompositions comprising 5-azacytidine, and optionally a permeationenhancer, wherein the formulations and compositions are prepared fororal administration. In a particular embodiment, the formulations andcompositions are prepared for release of 5-azacytidine substantially inthe stomach. In specific embodiments, 5-azacytidine and thepharmaceutical formulations and compositions are used for treatingdiseases and disorders associated with abnormal cell proliferation,wherein 5-azacytidine, the formulations and compositions are preparedfor oral administration, preferably for release of 5-azacytidinesubstantially in the stomach. Particular embodiments relate to the use5-azacytidine for the preparation of pharmaceutical formulations andcompositions for treating particular medical indications, as providedherein. The pharmaceutical formulations and compositions including5-azacytidine provided herein are intended for oral delivery of5-azacytidine to subjects in need thereof. Oral delivery formatsinclude, but are not limited to, tablets, capsules, caplets, solutions,suspensions, and syrups.

Particular embodiments herein provide solid oral dosage forms that aretablets or capsules. In certain embodiments, the formulation is a tabletcontaining 5-azacytidine. In certain embodiments, the formulation is acapsule containing 5-azacytidine. In certain embodiments, the tablets orcapsules provided herein optionally comprise one or more excipients,such as, for example, glidants, diluents, lubricants, colorants,disintegrants, granulating agents, binding agents, polymers, and coatingagents. In certain embodiments, embodiments herein encompass the use of5-azacytidine, for the preparation of a pharmaceutical composition fortreating a disease associated with abnormal cell proliferation, whereinthe composition is prepared for oral administration.

Pharmacokinetic Characteristics of Certain Dosage Forms Provided Herein

In certain embodiments, the formulations including 5-azacytidine effectan immediate release of the active pharmaceutical ingredient (API) uponoral administration. In particular embodiments, the formulationsincluding 5-azacytidine comprise a therapeutically effective amount of5-azacytidine (and, optionally, one or more excipients) and effect animmediate release of the API upon oral administration.

In certain embodiments, the formulations including 5-azacytidine releasethe API substantially in the stomach upon oral administration. Incertain embodiments, the formulations effect an immediate release of5-azacytidine upon oral administration. In certain embodiments, theformulations further comprise a drug release controlling component whichis capable of releasing 5-azacytidine substantially in the stomach. Incertain embodiments, the formulations optionally further comprises adrug release controlling component, wherein the drug release controllingcomponent is adjusted such that the release of 5-azacytidine occurssubstantially in the stomach. In particular embodiments, the drugrelease controlling component is adjusted such that the release of5-azacytidine is immediate and occurs substantially in the stomach. Inparticular embodiments, the drug release controlling component isadjusted such that the release of 5-azacytidine is sustained and occurssubstantially in the stomach. In certain embodiments, the formulation of5-azacytidine releases the API substantially in the stomach, and,subsequently, releases the remainder of the API in the intestine uponoral administration.

Methods by which skilled practitioners can assess the oralbioavailability of a drug formulation in a subject are known in the art.Such methods, include, for example, comparing various pharmacokineticparameters, such as, but not limited to, maximum plasma concentration(“Cmax”), time to maximum plasma concentration (“Tmax”), orarea-under-the-curve (“AUC”) determinations.

Particular embodiments herein provide pharmaceutical formulations (e.g.,immediate release oral formulations and/or formulations that release theAPI substantially in the stomach) including 5-azacytidine that achieve aparticular AUC value (e.g., AUC(0-t) or AUC(0-∞)) in the subject (e.g.,human) to which the formulation is orally administered. Particularembodiments provide oral formulations that achieve an AUC value of atleast 25 ng-hr/mL, at least 50 ng-hr/mL, at least 75 ng-hr/mL, at least100 ng-hr/mL, at least 150 ng-hr/mL, at least 200 ng-hr/mL, at least 250ng-hr/mL, at least 300 ng-hr/mL, at least 350 ng-hr/mL, at least 400ng-hr/mL, at least 450 ng-hr/mL, at least 500 ng-hr/mL, at least 550ng-hr/mL, at least 600 ng-hr/mL, at least 650 ng-hr/mL, at least 700ng-hr/mL, at least 750 ng-hr/mL, at least 800 ng-hr/mL, at least 850ng-hr/mL, at least 900 ng-hr/mL, at least 950 ng-hr/mL, at least 1000ng-hr/mL, at least 1100 ng-hr/mL, at least 1200 ng-hr/mL, at least 1300ng-hr/mL, at least 1400 ng-hr/mL, at least 1500 ng-hr/mL, at least 1600ng-hr/mL, at least 1700 ng-hr/mL, at least 1800 ng-hr/mL, at least 1900ng-hr/mL, at least 2000 ng-hr/mL, at least 2250 ng-hr/mL, or at least2500 ng-hr/mL. In particular embodiments, the AUC determination isobtained from a time-concentration pharmacokinetic profile obtained fromthe blood samples of human patients following dosing.

Particular embodiments herein provide pharmaceutical formulations (e.g.,immediate release oral formulations and/or formulations that release theAPI substantially in the stomach) including 5-azacytidine that achieve aparticular maximum plasma concentration (“Cmax”) in the subject to whichthe formulation is orally administered. Particular embodiments provideoral formulations that achieve a Cmax of 5-azacytidine of at least 25ng/mL, at least 50 ng/mL, at least 75 ng/mL, at least 100 ng/mL, atleast 150 ng/mL, at least 200 ng/mL, at least 250 ng/mL, at least 300ng/mL, at least 350 ng/mL, at least 400 ng/mL, at least 450 ng/mL, atleast 500 ng/mL, at least 550 ng/mL, at least 600 ng/mL, at least 650ng/mL, at least 700 ng/mL, at least 750 ng/mL, at least 800 ng/mL, atleast 850 ng/mL, at least 900 ng/mL, at least 950 ng/mL, at least 1000ng/mL, at least 1100 ng/mL, at least 1200 ng/mL, at least 1300 ng/mL, atleast 1400 ng/mL, at least 1500 ng/mL, at least 1600 ng/mL, at least1700 ng/mL, at least 1800 ng/mL, at least 1900 ng/mL, at least 2000ng/mL, at least 2250 ng/mL, or at least 2500 ng/mL. Particularembodiments herein provide pharmaceutical formulations (e.g., immediaterelease oral formulations and/or formulations that release the APIsubstantially in the stomach) including 5-azacytidine that achieve aparticular time to maximum plasma concentration (“Tmax”) in the subjectto which the formulation is orally administered. Particular embodimentsprovide oral formulations that achieve a Tmax of 5-azacytidine of lessthan 10 minutes, less than 15 minutes, less than 20 minutes, less than25 minutes, less than 30 minutes, less than 35 minutes, less than 40minutes, less than 45 minutes, less than 50 minutes, less than 55minutes, less than 60 minutes, less than 65 minutes, less than 70minutes, less than 75 minutes, less than 80 minutes, less than 85minutes, less than 90 minutes, less than 95 minutes, less than 100minutes, less than 105 minutes, less than 110 minutes, less than 115minutes, less than 120 minutes, less than 130 minutes, less than 140minutes, less than 150 minutes, less than 160 minutes, less than 170minutes, less than 180 minutes, less than 190 minutes, less than 200minutes, less than 210 minutes, less than 220 minutes, less than 230minutes, or less than 240 minutes In particular embodiments, the Tmaxvalue is measured from the time at which the formulation is orallyadministered.

Design of Certain Dosage Forms Provided Herein

Provided herein are dosage forms designed to maximize the absorptionand/or efficacious delivery of 5-azacytidine, upon oral administration,e.g., for release substantially in the stomach. Accordingly, certainembodiments herein provide a solid oral dosage form of 5-azacytidine,such as, for example, 5-azacytidine, using pharmaceutical excipientsthat effect immediate release of the API upon oral administration, e.g.,substantially in the stomach. Particular immediate release formulationscomprise a specific amount of 5-azacytidine and optionally one or moreexcipients. In certain embodiments, the formulation is an immediaterelease tablet or an immediate release capsule (such as, e.g., an HPMCcapsule).

Provided herein are methods of making the formulations provided hereinincluding 5-azacytidine provided herein (e.g., immediate release oralformulations and/or formulations that release the API substantially inthe stomach). In particular embodiments, the formulations providedherein are prepared using conventional methods known to those skilled inthe field of pharmaceutical formulation, as described, e.g., inpertinent textbooks. See, e.g., REMINGTON, THE SCIENCE AND PRACTICE OFPHARMACY, 20th Edition, Lippincott Williams & Wilkins, (2000); ANSEL etal., PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 7th Edition,Lippincott Williams & Wilkins, (1999); GIBSON, PHARMACEUTICALPREFORMULATION AND FORMULATION, CRC Press (2001).

In certain embodiments, the formulation is a tablet, wherein the tabletis manufactured using standard, art-recognized tablet processingprocedures and equipment. In certain embodiments, the method for formingthe tablets is direct compression of a powdered, crystalline and/orgranular composition including 5-azacytidine, alone or in combinationwith one or more excipients, such as, for example, carriers, additives,polymers, or the like. In certain embodiments, as an alternative todirect compression, the tablets are prepared using wet granulation ordry granulation processes. In certain embodiments, the tablets aremolded rather than compressed, starting with a moist or otherwisetractable material. In certain embodiments, compression and granulationtechniques are used.

In certain embodiments, the compressed tablet of 5-azacytidine isfilm-coated. In some embodiments, the film-coated tablets are compressedtablets coated with a thin layer of a polymer capable of forming askin-like film over the tablet. The film is usually colored and has theadvantage to be more durable, less bulky, and less time-consuming toapply. By its composition, the coating may be designed to rupture andexpose the core tablet at the desired location within thegastrointestinal tract. The film-coating process, which places a thinskin-tight coating of a plastic-like material over the compressedtablet, may produce coated tablets having essentially the same weight,shape, and size as the originally compressed tablet. In someembodiments, the film-coating is colored to make the tablets attractiveand distinctive. In some embodiments, the film-coating solutions arenon-aqueous or aqueous. In particular embodiments, the non-aqueoussolutions are optionally contain one or more of the following types ofmaterials to provide the desired coating to the tablets: (1) a filmformer capable of producing smooth, thin films reproducible underconventional coating conditions and applicable to a variety of tabletshapes, such as, for example, cellulose acetate phthalate; (2) analloying substance providing water solubility or permeability to thefilm to ensure penetration by body fluids and therapeutic availabilityof the drug, such as, for example, polyethylene glycol; (3) aplasticizer to produce flexibility and elasticity of the coating andthus provide durability, such as, for example, castor oil; (4) asurfactant to enhance spreadability of the film during application, suchas, for example, polyoxyethylene sorbitan derivatives; (5) opaquants andcolorants to make the appearance of the coated tablets attractive anddistinctive, such as, for example, titanium dioxide as an opaquant, andFD&C or D&C dyes as a colorant; (6) sweeteners, flavors, or aromas toenhance the acceptability of the tablet to the subject, such as, forexample, saccharin as sweeteners, and vanillin as flavors and aromas;(7) a glossant to provide a luster to the tablets without a separatepolishing operation, such as, for example, beeswax; and (8) a volatilesolvent to allow the spread of the other components over the tabletswhile allowing rapid evaporation to permit an effective yet speedyoperation, such as, for example, alcohol-acetone mixture. In certainembodiments, an aqueous film-coating formulation contains one or more ofthe following: (1) film-forming polymer, such as, for example, celluloseether polymers as hydroxypropyl methyl-cellulose, hydroxypropylcellulose, and methyl-cellulose; (2) plasticizer, such as, for example,glycerin, propylene glycol, polyethylene glycol, diethyl phthalate, anddibutyl subacetate; (3) colorant and opacifier, such as, for example,FD&C or D&C lakes and iron oxide pigments; or (4) vehicle, such as, forexample, water.

In certain embodiments, the pharmaceutical formulation is an immediaterelease tablet of 5-azacytidine. In certain embodiments, the immediaterelease tablet is designed, e.g., to disintegrate and release the APIabsent of any special rate-controlling features, such as specialcoatings and other techniques.

In certain embodiments, the pharmaceutical formulations provided hereincontain 5-azacytidine and, optionally, one or more excipients to form a“drug core.” Optional excipients include, e.g., diluents (bulkingagents), lubricants, disintegrants, fillers, stabilizers, surfactants,preservatives, coloring agents, flavoring agents, binding agents,excipient supports, glidants, permeation enhancement excipients,plasticizers and the like, e.g., as known in the art.

One or more diluents may be used, e.g., to increase bulk so that apractical size tablet is ultimately provided. Diluents also include,e.g., ammonium alginate, calcium carbonate, calcium phosphate, calciumsulfate, cellulose acetate, compressible sugar, confectioner's sugar,dextrates, dextrin, dextrose, erythritol, ethylcellulose, fructose,fumaric acid, glyceryl palmitostearate, isomalt, kaolin, lacitol,lactose, mannitol, magnesium carbonate, magnesium oxide, maltodextrin,maltose, medium-chain triglycerides, microcrystalline cellulose,microcrystalline silicified cellulose, powered cellulose, polydextrose,polymethylacrylates, simethicone, sodium alginate, sodium chloride,sorbitol, starch, pregelatinized starch, sucrose,sulfobutylether-β-cyclodextrin, talc, tragacanth, trehalose, andxylitol. In some embodiments, the diluents comprise mannitol andmicrocrystalline silicified cellulose. Diluents may be used in amountscalculated to obtain a desired volume for a tablet. In some embodiments,a diluent is used in an amount of about 5% or more, about 10% or more,about 15% or more, about 20% or more, about 22% or more, about 24% ormore, about 26% or more, about 28% or more, about 30% or more, about 32%or more, about 34% or more, about 36% or more, about 38% or more, about40% or more, about 42% or more, about 44% or more, about 46% or more,about 48% or more, about 50% or more. In some embodiments, a diluentused in the formulation is between about 20% and about 40% w/w of thedrug core.

One or more lubricants may be used, e.g., to facilitate tabletmanufacture. Examples of suitable lubricants include, for example,vegetable oils such as peanut oil, cottonseed oil, sesame oil, oliveoil, corn oil, and oil of theobroma, glycerin, magnesium stearate,calcium stearate, and stearic acid. In certain embodiments, stearates,if present, represent no more than approximately 2 weight % of thedrug-containing core. In particular embodiments, the lubricant ismagnesium stearate. In certain embodiments, the lubricant is present,relative to the drug core, in an amount of about 0.2% w/w of the drugcore, about 0.4% w/w of the drug core, about 0.6% w/w of the drug core,about 0.8% w/w of the drug core, about 1.0% w/w of the drug core, about1.2% w/w of the drug core, about 1.4% w/w of the drug core, about 1.6%w/w of the drug core, about 1.8% w/w of the drug core, about 2.0% w/w ofthe drug core, about 2.2% w/w of the drug core, about 2.4% w/w of thedrug core, about 2.6% w/w of the drug core, about 2.8% w/w of the drugcore, about 3.0% w/w of the drug core, about 3.5% w/w of the drug core,about 4% w/w of the drug core, about 4.5% w/w of the drug core, or about5% w/w of the drug core. In some embodiments, the lubricant is presentin an amount of between about 0.5% and about 5% w/w of the drug core, orbetween about 1% and about 3% w/w of the drug core.

One or more disintegrants may be used, e.g., to facilitatedisintegration of the tablet, and may be, e.g., starches, clays,celluloses, algins, gums or crosslinked polymers. Disintegrants alsoinclude, e.g., alginic acid, carboxymethylcellulose calcium,carboxymethylcellulose sodium (e.g., AC-DI-SOL, PRIMELLOSE), colloidalsilicon dioxide, croscarmellose sodium, crospovidone (e.g., KOLLIDON,POLYPLASDONE), guar gum, magnesium aluminum silicate, methyl cellulose,microcrystalline cellulose, polacrilin potassium, powdered cellulose,pregelatinized starch, sodium alginate, sodium starch glycolate (e.g.,EXPLOTAB) and starch. In some embodiments, the disintegrant iscroscarmellose sodium. In certain embodiments, the disintegrant is,relative to the drug core, present in the amount of about 1% w/w of thedrug core, about 2% w/w of the drug core, about 3% w/w of the drug core,about 4% w/w of the drug core, about 5% w/w of the drug core, about 6%w/w of the drug core, about 7% w/w of the drug core, about 8% w/w of thedrug core, about 9% w/w of the drug core, or about 10% w/w of the drugcore. In some embodiments, the disintegrant is present in the amount ofabout between about 1% and about 10% w/w of the drug core, between about2% and about 8% w/w of the drug core.

B. 5-Azacytidine with at Least One Additional Therapeutic Agent

In particular embodiments, 5-azacytidine compositions provided hereinfurther comprise one, two, three, or more other pharmacologically activesubstances (also termed herein “additional therapeutic agents,” “secondactive agents,” or the like). In some embodiments, the 5-azacytidinecompositions are oral formulations. In some embodiments, the5-azacytidine oral compositions with at least one additional therapeuticagent is used for treating any of the diseases or disorders disclosedherein. In particular embodiments, the oral formulations provided hereincomprise the additional therapeutic agent(s) in a therapeuticallyeffective amount. In particular embodiments, 5-azacytidine and theadditional therapeutic agent(s) are co-formulated together in the samedosage form using methods of co-formulating active pharmaceuticalingredients, including methods disclosed herein and methods known in theart. In other embodiments, 5-azacytidine and the additional therapeuticagent(s) are co-administered in separate dosage forms. In someembodiments, certain combinations work synergistically in the treatmentof particular diseases or disorders, including, e.g., types of cancerand certain diseases and conditions associated with, or characterizedby, undesired angiogenesis or abnormal cell proliferation.

Examples of additional therapeutic agents include but are not limited togilteritinib, midostaurin, quizartinib, enasidenib, ivosidenib, andvenetoclax.

Examples of additional therapeutic agents include but are not limited toFLT3 inhibitors, IDH2 inhibitors, IDH1 inhibitors, and BCL2 inhibitors.Examples of first generation FLT3 inhibitors include but are not limitedto midostaurin, lestaurtinib, sunitinib (Sutent®), and sorafenib(Nexavar®). Examples of second generation FLT3 inhibitors include butare not limited to quizartinib, crenolanib, pexidartinib (PLX3397), andgilteritinib (ASP2215), are more potent and selective than thefirst-generation inhibitors. Examples of IDH inhibitors, including IDH1and/or IDH2 inhibitors, include but are not limited to ivosidenib andenasidenib. Examples of BCL2 inhibitors include but are not limited tovenetoclax (ABT-199), navitoclax (ABT-263), ABT-737(4-[4-[[2-(4-chlorophenyl)phenyl]methyl]piperazin-1-yl]-N-[4-[[(2R)-4-(dimethylamino)-1-phenylsulfanylbutan-2-yl]amino]-3-nitrophenyl]sulfonylbenzamide),obatoclax mesylate (GX15-070), TW-37(N-[4-(2-tert-butylphenyl)sulfonylphenyl]-2,3,4-trihydroxy-5-[(2-propan-2-ylphenyl)methyl]benzamide),AT101 ((R)-(−)-Gossypol), HA14-1(2-Amino-6-bromo-α-cyano-3-(ethoxycarbonyl)-4H-1-benzopyran-4-aceticacid ethyl ester), and sabutoclax.

C. Combination of 5-Azacytidine with Venetoclax as the at Least OneAdditional Therapeutic Agent Surprisingly Safe and Effective forTreating Diseases

In some embodiments, an oral pharmaceutical composition comprising5-azacytidine is used with venetoclax as the additional therapeuticagent. In some embodiments, the 5-azacytidine oral compositions is usedwith venetoclax for safely and effectively treating any of the diseasesor disorders disclosed herein.

Venetoclax is a selective inhibitor of BCL-2 and is marketed asVENCLEXTA™, which is in the form of a tablet. Venetoclax is indicated inthe US: (i) for the treatment of adult patients with chronic lymphocyticleukemia (CLL) or small lymphocytic lymphoma (SLL); (ii) in combinationwith injectable 5-azacytidine or decitabine or low-dose cytarabine forthe treatment of newly-diagnosed acute myeloid leukemia (AML) in adultswho are age 75 years or older, or who have comorbidities that precludeuse of intensive induction chemotherapy. In some embodiments, patientsreceive 20 mg/m² of cytarabine once a day subcutaneously for 10consecutive days every 4 weeks as the low-dose cytarabine.

Therapy with venetoclax is initiated according to a weekly ramp-upschedule over a specific period of several days or weeks to therecommended daily dose. For treating CLL and SLL, venetoclax is atadministered at a daily dose of 20 mg for Week 1, a daily dose of 50 mgfor Week 2, a daily dose of 100 mg for Week 3, a daily dose of 200 mgfor Week 4, and a daily dose of 400 mg for Week 5 and beyond. Fortreating AML in combination therapy with another agent, such asinjectable 5-azacytidine, venetoclax is at administered at a daily doseof 100 mg for Day 1, a daily dose of 200 mg for Day 2, and a daily doseof 400 mg for Days 3 and beyond. VIDAZA® (5-azacytidine for injection)is administered in 28-day cycles, beginning on Day 1 of venetoclaxtreatment, at a dosage of 75 mg/m², IV or subcutaneously, on Days 1-7 ofeach cycle.

In some embodiments, venetoclax is administered orally. In someembodiments, the venetoclax is administered in a form of a tablet. Insome embodiments, venetoclax is administered daily. In some embodiments,venetoclax is administered at a dose of from about 20 mg to about 400mg, such as about 20 mg, about 50 mg, about 100 mg, about 200 mg, orabout 400 mg. In some embodiments, venetoclax is administered at a doseof about 400 mg.

In some embodiments, 5-azacytidine and venetoclax are administeredconcomitantly. In some embodiments, 5-azacytidine and venetoclax areadministered sequentially. In some embodiments, where the 5-azacytidineand venetoclax are administered sequentially, the 5-azacytidine isadministered first. In some embodiments, 5-azacytidine and venetoclaxare administered as separate dosage forms, such as injections suitablefor intravenous or subcutaneous use and/or tablets or capsules for oraluse. In some embodiments, 5-azacytidine and venetoclax are co-formulatedas a single unit dosage form, such as an injection suitable forintravenous or subcutaneous use or a tablet or capsule for oral use.

D. Combination of 5-Azacytidine with Gilteritinib as the at Least OneAdditional Therapeutic Agent Surprisingly Safe and Effective forTreating Diseases

In some embodiments, an oral pharmaceutical composition comprising5-azacytidine is used with gilteritinib as the additional therapeuticagent. In some embodiments, the 5-azacytidine oral compositions is usedwith gilteritinib for safely and effectively treating any of thediseases or disorders disclosed herein.

Gilteritinib is a tyrosine kinase inhibitor and is marketed as XOSPATA®,which is in the form of a tablet. Gilteritinib is indicated in the USfor the treatment of adult patients who have relapsed or refractoryacute myeloid leukemia (AML) with a FLT3 mutation as detected by anFDA-approved test. The recommended starting dose for gilteritinib is 120mg orally once daily with or without food.

In some embodiments, the gilteritinib is administered orally. In someembodiments, the gilteritinib is administered in a form of a tablet. Insome embodiments, the gilteritinib is administered daily. In someembodiments, the gilteritinib is administered at a dose of from about 20mg to about 400 mg, from about 40 mg to about 400 mg, from about 40 mgto about 200 mg, such as about 20 mg, about 40 mg, about 50 mg, about 80mg, about 100 mg, about 120 mg, about 160 mg, about 200 mg, or about 400mg. In some embodiments, the gilteritinib is administered at a dose ofabout 120 mg.

In some embodiments, 5-azacytidine and gilteritinib are administeredconcomitantly. In some embodiments, 5-azacytidine and gilteritinib areadministered sequentially. In some embodiments, where the 5-azacytidineand gilteritinib are administered sequentially, the 5-azacytidine isadministered first. In some embodiments, 5-azacytidine and gilteritinibare administered as separate dosage forms, such as injections suitablefor intravenous or subcutaneous use and/or tablets or capsules for oraluse. In some embodiments, 5-azacytidine and gilteritinib areco-formulated as a single unit dosage form, such as an injectionsuitable for intravenous or subcutaneous use or a tablet or capsule fororal use.

E. Methods of Use for 5-Azacytidine and at Least One AdditionalTherapeutic Agent

As described herein, certain embodiments herein provide methods oftreating a subject having acute myeloid leukemia (AML), wherein themethod includes administering to the subject (i) a pharmaceuticalcomposition comprising 5-azacytidine; and (ii) at least one additionaltherapeutic agent. In some embodiments, the pharmaceutical compositioncomprising 5-azacytidine is administered orally. Also in someembodiments, the pharmaceutical composition comprising 5-azacytidine asdescribed herein is used with at least one additional therapeutic agentare used for treating AML in a subject, including a human patient.

In some embodiments, 5-azacytidine and one or more therapeutic agentsare co-administered to subjects to yield a synergistic therapeuticeffect. The co-administered agent may be a cancer therapeutic agentdosed orally or by injection.

In certain embodiments, methods provided herein for treating disordersrelated to abnormal cell proliferation comprise orally administering aformulation comprising a therapeutically effective amount of5-azacytidine. Particular therapeutic indications relating to themethods provided herein are disclosed herein. In certain embodiments,the therapeutically effective amount of 5-azacytidine in thepharmaceutical formulation is an amount as disclosed herein. In certainembodiments, the precise therapeutically effective amount of5-azacytidine in the pharmaceutical formulation will vary depending on,e.g., the age, weight, disease and/or condition of the subject.

In particular embodiments, the disorders related to abnormal cellproliferation include, but are not limited to, myelodysplastic syndrome(MDS), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL),chronic myeloid leukemia (CIVIL), leukemia, chronic lymphocytic leukemia(CLL), lymphoma (including non-Hodgkin's lymphoma (NHL) and Hodgkin'slymphoma), multiple myeloma (MM), sarcoma, melanoma, carcinoma,adenocarcinoma, chordoma, breast cancer, colorectal cancer, ovariancancer, lung cancer (e.g., non-small-cell lung cancer and small-celllung cancer), testicular cancer, renal cancer, pancreatic cancer, bonecancer, gastric cancer, head and neck cancer, and prostate cancer. Inparticular embodiments, the disorder related to abnormal cellproliferation is lymphoma. In particular embodiments, the lymphoma isangioimmunoblastic T-cell lymphoma. In particular embodiments, thedisorder related to abnormal cell proliferation is MDS. In particularembodiments, the disorder related to abnormal cell proliferation is AML.

Particular embodiments herein provide methods for treating a subjecthaving a disease or disorder provided herein by orally administering apharmaceutical composition provided herein, wherein the treatmentresults in improved survival of the patient. In certain embodiments, theimproved survival is measured as compared to one or more standard careregimens. Particular embodiments herein provide methods for treating asubject having a disease or disorder provided herein by orallyadministering a pharmaceutical composition provided herein, wherein thetreatment provides improved effectiveness for treating the disease ordisorder. In particular embodiments, the improved effectiveness ismeasured using one or more endpoints for cancer clinical trials, asrecommended by the U.S. Food and Drug Administration (FDA). For example,FDA provides Guidance for Industry on Clinical Trial Endpoints for theApproval of Cancer Drugs and Biologics(http://www.fda.gov/CbER/gdlns/clintrialend.htm). The FDA endpointsinclude, but are not limited to, Overall Survival, Endpoints Based onTumor Assessments such as (i) Disease-Free Survival (ii) ObjectiveResponse Rate, (iii) Time to Progression and Progression-Free Survivaland (iv) Time-to-Treatment Failure. Endpoints Involving SymptomEndpoints may include Specific Symptom Endpoints such as (i) Time toprogression of cancer symptoms and (ii) A composite symptom endpoint.Biomarkers assayed from blood or body fluids may also be useful todetermine the management of the disease. In some embodiments, theimprovement can be about 5%, about 10%, about 15%, about 20%, about 25%,about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,about 95%, or about 100%.

Subjects in need of treatment can be members of a patient populationwith an increased risk of AML. For example, several inherited geneticdisorders and immunodeficiency states are associated with an increasedrisk of AML. These include disorders with defects in DNA stability,leading to random chromosomal breakage, such as Bloom's syndrome,Fanconi's anemia, Li-Fraumeni kindreds, ataxia-telangiectasia, andX-linked agammaglobulinemia.

In certain embodiments, methods provided herein comprise treating acutepromyelocytic leukaemia (APML) by administering a pharmaceuticalcomposition comprising 5-azacytidine in combination with one or moreadditional agents to a subject in need thereof. APML is a rare sub-typeof AML and is sometimes referred to as AML M31. This subtype ischaracterized by promyelocytic blasts containing the 15; 17 chromosomaltranslocation. This translocation leads to the generation of the fusiontranscript comprised of the retinoic acid receptor and a sequence PML.

In some embodiments, methods described herein are used to treat specifictypes of acute myeloid leukemia. Illustrative types of acute myeloidleukemia include but are not limited to, acute myeloid leukemia withrecurrent genetic abnormalities, acute myeloid leukemia withmyelodysplasia-related changes, therapy-related myeloid neoplasms,myeloid sarcoma, myeloid proliferations related to Down syndrome,blastic plasmacytoid dendritic cell neoplasm, and/or acute promyelocyticleukaemia.

In some embodiments, the AML is characterized as caused by any one ofthe following mutations: Fms-related tyrosine kinase 3 (FLT3), Kirstenrat sarcoma viral oncogene homolog (KRAS), neuroblastoma RAS viral(V-Ras) oncogene homolog (NRAS), proto-oncogene c-Kit (KIT), proteintyrosine phosphatase non-receptor type 11 (PTPN11), neurofibromin 1(NF1), DNA methyltransferase 3A (DNMT3A), isocitrate dehydrogenase 1(IDH1), isocitrate dehydrogenase 2 (IDH2), ten-eleven translocation-2(TET2), additional sex comb-like 1 (ASXL1), enhancer of zeste homolog 2(EZH2), mixed-lineage leukemia 1/histone-lysine N-methyltransferase 2A(MLL/KMT2A), nucleophosmin (NPM1), CCAAT enhancer binding protein alpha(CEBPA), runt-related transcription factor 1 (RUNX1), GATA-bindingfactor 2 (GATA2), tumor protein p53 (TP53), serine and arginine richsplicing factor 2 (SRSF2), U2 small nuclear RNA auxiliary factor 1(U2AF1), splicing factor 3b subunit 1 (SF3B1), zinc finger (CCCH type),RNA-binding motif and serine/arginine rich 2 (ZRSR2), RAD21 cohesincomplex component (RAD21), stromal antigen 1 (STAG1), stromal antigen 2(STAG2), structural maintenance of chromosomes 1A (SMC1A), andstructural maintenance of chromosomes protein 3 (SMC3).

In some embodiments, the AML is characterized as caused by a FLT3-ITDmutation. In some embodiments, the AML is resistant to treatment withthe at least one additional therapeutic agent alone. In someembodiments, the 5-azacytidine is administered before the at least oneadditional therapeutic agent. In some embodiments, the AML is responsiveto treatment with a FLT3 inhibitor. In some embodiments, the AML ischaracterized as having an overexpression of MCL-1.

In some embodiments, the 5-azacytidine primes the cancer cells forapoptosis mediated by the at least one additional therapeutic agent bydownregulating the expression of MCL-1. In some embodiments,downregulating the expression of MCL-1 is mediated by caspase-dependentand independent mechanisms. In some embodiments, the 5-azacytidine andat least one additional therapeutic agent augments MCL-1 degradation.

In certain embodiments, methods provided herein comprise treatinglymphoma by administering a pharmaceutical composition comprising5-azacytidine in combination with one or more additional agents to asubject in need thereof. Types of lymphomas include non-Hodgkin lymphomaand Hodgkin's disease. Examples of lymphoma include, but are not limitedto, diffuse large B-cell lymphoma, anaplastic large-cell lymphoma,Burkitt lymphoma, lymphoblastic lymphoma, mantle cell lymphoma,peripheral T-cell lymphoma, follicular lymphoma, cutaneous T-celllymphoma, lymphoplasmacytic lymphoma, marginal zone B-cell lymphoma,MALT lymphoma, small-cell lymphocytic lymphoma, and angioimmunoblasticT-cell lymphoma. In some embodiments, the lymphoma is angioimmunoblasticT-cell lymphoma.

In certain embodiments, methods provided herein comprise treatingmyelodysplastic syndromes, by administering a pharmaceutical compositioncomprising 5-azacytidine in combination with one or more additionalagents to a subject in need thereof. MDS may also be classified by usingthe Revised International Prognostic Scoring System (IPSS-R), whichclassifies patients into 1 of 5 groups, from very low risk to very highrisk, based on risk of mortality and transformation to acute myeloidleukemia (AML). Higher-risk MDS as used in the disclosure is defined asHigh or Very High risk according to the Revised International ScoringSystem (IPSS-R). Greenberg, P. L. et al., Blood 2012 Sep. 20; 120 (12):2454-2465. The scoring system for the IPSS-R is based on the followingfactors: the percentage of blasts (very early forms of blood cells) inthe bone marrow, the type and number of chromosome abnormalities in thecells, the level of red blood cells (measured as hemoglobin) in thepatient's blood, the level of platelets in the patient's blood, and thelevel of neutrophils (a type of white blood cell) in the patient'sblood. Each factor is assigned a score and the total sum of the score isused to assign the MDS patient into one of the following five riskgroups: Very low (Risk score of ≤1.5); Low risk (Risk score of >1.5-3);Intermediate risk (Risk score of >3-4.5); High risk (Risk Scoreof >4.5-6); and Very high risk (Risk Score of >6). About 13% of MDSpatients are classified as High Risk, which has a mean overall survivalof 1.6 years while about 10% of MDS patients are classified as Very HighRisk, which has a mean overall survival of 0.8 years. In someembodiments, the MDS is MDS that is classified as High Risk or Very HighRisk as defined by the IPSS-R.

F. Dosing Regimens for 5-Azacytidine and an Additional Therapeutic Agent

Certain embodiments herein provide methods of treating diseases ordisorders disclosed herein (e.g., diseases or disorders involvingabnormal cell proliferation), wherein the methods compriseco-administering an oral formulation disclosed herein (such as, forexample, an oral formulation comprising 5-azacytidine) with one or moreadditional therapeutic agents (such as, for example, a cancertherapeutic agent) to yield a synergistic therapeutic effect. Particularco-administered therapeutic agents useful in the methods disclosedherein are disclosed throughout the specification. In particularembodiments, the additional therapeutic agent is co-administered in anamount that is a therapeutically effective amount. In particularembodiments, the additional therapeutic agent is co-administered in aseparate dosage form from 5-azacytidine dosage form with which it isco-administered. In particular embodiments, the additional therapeuticagent is co-administered in a dosage form (e.g., a single unit dosageform) together with 5-azacytidine with which it is co-administered. Insuch cases, 5-azacytidine (e.g., azacitidine) and the additionaltherapeutic agent may be co-formulated together in the same dosage formusing methods of co-formulating active pharmaceutical ingredients,including methods disclosed herein and methods known in the art.

In some embodiments provided herein is a method of treating a humanhaving acute myeloid leukemia, wherein the method includes administeringto the human a pharmaceutical composition including 5-azacytidine; andwherein the method further includes administering at least oneadditional therapeutic agent.

In some embodiments provided herein is a method of treating a humanhaving myelodysplastic syndrome, wherein the method includesadministering to the human a pharmaceutical composition including5-azacytidine; and wherein the method further includes administering atleast one additional therapeutic agent.

In some embodiments provided herein, the additional therapeutic agent isselected from gilteritinib, midostaurin, quizartinib, enasidenib,ivosidenib, or venetoclax.

In some embodiments provided herein, the additional therapeutic agent isa FLT3 inhibitor. In one embodiment, the FLT3 inhibitor is gilteritinib,midostaurin, or quizartinib.

In some embodiments provided herein, the additional therapeutic agent isan IDH2 inhibitor. In one embodiment, the IDH2 inhibitor is enasidenib.

In some embodiments provided herein, the additional therapeutic agent isan IDH1 inhibitor. In one embodiment, the IDH1 inhibitor is ivosidenib.

In some embodiments provided herein, the additional therapeutic agent isa Bcl2 inhibitor. In one embodiment, the Bcl2 inhibitor is venetoclax.

In one embodiment provided herein, the pharmaceutical composition thatincludes 5-azacytidine is administered orally.

In one embodiment provided herein, the pharmaceutical compositionincluding 5-azacytidine is a capsule.

In one embodiment provided herein, the pharmaceutical compositionincluding 5-azacytidine is a tablet.

In some embodiments, the 5-azacytidine and the at least one additionaltherapeutic agent are administered concomitantly. In some embodiments,the 5-azacytidine and the at least one additional therapeutic agent areadministered sequentially wherein the 5-azacytidine is administeredfirst. In some embodiments, 5-azacytidine and the at least oneadditional therapeutic agent are co-formulated as a single unit dosageform. In some embodiments, the additional therapeutic agent isadministered parenterally. In some embodiments, the additionaltherapeutic agent is administered orally.

In some embodiments, the 5-azacytidine is administered orally. In someembodiments, the 5-azacytidine is administered at a dose of about 50 mg,about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about400 mg, about 450 mg, about 500 mg, about 550 mg, or 600 mg orally. Insome embodiments, the 5-azacytidine is administered at a dose of about200 mg. In some embodiments, the 5-azacytidine is administered at a doseof about 300 mg. In some embodiments, 5-azacytidine is administeredorally for the first seven, fourteen, or twenty-one days of a cycle. Insome embodiments, the 5-azacytidine administered to the subject once ortwo times per day. In some embodiments, the 5-azacytidine isadministered in the form of a capsule or a tablet. In some embodiments,the tablet is a non-enteric coated tablet.

In some embodiments, the at least one additional therapeutic agent isgilteritinib. In some embodiments, the at least one additionaltherapeutic agent is midostaurin. In some embodiments, the at least oneadditional therapeutic agent is venetoclax.

In some embodiments, the 5-azacytidine and the at least one additionaltherapeutic agent provides a synergistic effect to treat the diseasesdisclosed herein. Synergy may be measured by using the highest singleagent (HSA) model and Combenefit package (Di Veroli et al.,Bioinformatics. 2016 Sep. 15; 32(18):2866-8.) A negative cell line isused as a control to determine whether there was a shift in EC₅₀ and/oran augmentation of the maximal inhibitory effect. In other words, theEC₅₀ and maximal inhibitory effect from the negative control cell lineprovide baseline potency results, and the shift in EC₅₀ and maximalinhibitory effect of the drug combination is compared to the resultsfrom the negative control cell line to determine whether the drugcombination provided a synergistic effect. Specifically, the followingsteps are used to determine the synergistic interactions between twodrugs: (a) a demonstration of shift in dose response curves determinedfrom their EC₅₀ (i.e., a potency shift) and/or an augmentation of themaximal inhibitory effect compared to the results from the negativecontrol cell line; (b) response surface analyses to visualize synergy,additivity or antagonism over a matrix of concentration between the twodrugs; and (c) analyzing the combination index score (derived using asoftware application Combenefit). The limit of where the synergy indexbecomes significant (such that the drug combination exhibits synergisticeffects) is determined empirically and is based on the variance in thedata and a confirmation in a potency shift in EC₅₀. In other words, acombination index, without the clear shift in dose response curves wouldnot constitute a synergistic interaction. As used herein, in someembodiments, the synergistic effect is defined as having an EC₅₀ shiftat about greater than about 4 and/or a synergy index of greater thanabout 20 as measured by the HSA model and Combenefit package. In someembodiments, the synergistic effect is defined as having an EC₅₀ shiftat about greater than 4 and/or a synergy index of greater than 20 asmeasured by the HSA model and Combenefit package.

In some embodiments, the 5-azacytidine primes the cancer cells forapoptosis mediated by the at least one additional therapeutic agent bydownregulating the expression of MCL-1. In some embodiments,downregulating the expression of MCL-1 is mediated by caspase-dependentand independent mechanisms. In some embodiments, the 5-azacytidine andat least one additional therapeutic agent augments MCL-1 degradation.

In some embodiments, the 5-azacytidine alters cell metabolism. In someembodiments, the 5-azacytidine causes cell cycle arrest. In someembodiments, the 5-azacytidine suppresses oxidative phosphorylation. Insome embodiments, the 5-azacytidine increases expression of ATF3(activating transcription factor 3). In some embodiments, the5-azacytidine decreases expression of SCD (stearoyl-CoA desaturase).

In some embodiments, the therapeutic effect of (1) 5-azacytidineadministered orally and at least one additional therapeutic agent isbetter than the therapeutic effect of (2) the 5-azacytidine alone, (3)the at least one additional therapeutic alone, and/or (4) thecombination of the 5-azacytidine administered intravenously orsubcutaneously and at the at least one additional therapeutic agent.

In some embodiments, the 5-azacytidine and at least one additionaltherapeutic agent increases median survival as compared to the5-azacytidine alone. In some embodiments, the 5-azacytidine and at leastone additional therapeutic agent increases median survival as comparedto the 5-azacytidine alone by about 10%, about 15%, about 20%, about25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%,about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about90%, about 95%, or about 100%, as measured by any clinically recognizedtechnique.

In some embodiments, the 5-azacytidine and venetoclax increases mediansurvival as compared to the 5-azacytidine alone. In some embodiments,the 5-azacytidine and venetoclax increases median survival as comparedto the 5-azacytidine alone by about 10%, about 15%, about 20%, about25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%,about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about90%, about 95%, or about 100%, as measured by any clinically recognizedtechnique.

In some embodiments, the 5-azacytidine and at least one additionaltherapeutic agent increases median survival as compared to at least oneadditional therapeutic agent alone. In some embodiments, the5-azacytidine and at least one additional therapeutic agent increasesmedian survival as compared to at least one additional therapeutic agentalone by about 10%, about 15%, about 20%, about 25%, about 30%, about35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%,about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, orabout 100%, as measured by any clinically recognized technique.

In some embodiments, the 5-azacytidine and venetoclax increases mediansurvival as compared to venetoclax alone. In some embodiments, the5-azacytidine and venetoclax increases median survival as compared tovenetoclax alone by about 10%, about 15%, about 20%, about 25%, about30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%,about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about95%, or about 100%, as measured by any clinically recognized technique.

In some embodiments, the 5-azacytidine and at least one additionaltherapeutic agent increases median survival as compared to 5-azacytidineadministered intravenously or subcutaneously and at least one additionaltherapeutic agent. In some embodiments, the 5-azacytidine and at leastone additional therapeutic agent increases median survival as comparedto 5-azacytidine administered intravenously or subcutaneously and atleast one additional therapeutic agent by about 10%, about 15%, about20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%,about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about85%, about 90%, about 95%, or about 100%, as measured by any clinicallyrecognized technique.

In some embodiments, the 5-azacytidine and venetoclax increases mediansurvival as compared to 5-azacytidine administered intravenously orsubcutaneously and venetoclax. In some embodiments, the 5-azacytidinevenetoclax increases median survival as compared to 5-azacytidineadministered intravenously or subcutaneously and venetoclax by about10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%,about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about75%, about 80%, about 85%, about 90%, about 95%, or about 100%, asmeasured by any clinically recognized technique.

In one embodiment provided herein, the method includes: (i)administering 5-azacytidine to the subject for 1, 2, or 3 days; and (ii)administering the at least one additional therapeutic agent to thesubject for one or more days. In one embodiment provided herein, themethod further includes repeating steps (i) and (ii).

In one embodiment provided herein, the method includes: (i)administering 5-azacytidine daily to the subject for 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, or 14 days; (ii) administering the at least oneadditional therapeutic agent to the subject for one or more days; and(iii) optionally repeating steps (i) and (ii).

In one embodiment provided herein, the method includes: (i)administering 5-azacytidine daily to the subject for 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days; and(ii) administering the at least one additional therapeutic agent to thesubject for one or more days. In one embodiment provided herein, themethod further includes repeating steps (i) and (ii).

In one embodiment provided herein, the method includes: (i)administering 5-azacytidine daily to the subject for 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, or 14 days of a 28-day cycle; (ii) concurrentlyadministering the at least one additional therapeutic agent daily to thesubject for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days of a 28-day cycle;and (iii) optionally repeating steps (i) and (ii).

In one embodiment provided herein, the method includes: (i)administering 5-azacytidine daily to the subject for 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days of a28-day cycle; (ii) concurrently administering the at least oneadditional therapeutic agent daily to the subject for 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, or 28 days of a 28-day cycle; and (iii) optionally repeatingsteps (i) and (ii).

In one embodiment provided herein, the method includes the sequentialsteps of: (i) administering 5-azacytidine to the subject for 7 days of a28-day cycle; (ii) administering the at least one additional therapeuticagent to the subject for 1 day of a 28-day cycle; (iii) administering5-azacytidine to the subject for 6 days of a 28-day cycle; and (iv)repeating steps (i) to (iii) after 7 days of a resting period.

In one embodiment provided herein, the method includes the sequentialsteps of: (i) administering 5-azacytidine daily to the subject for 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21days of a 28-day cycle; (ii) administering the at least one additionaltherapeutic agent daily to the subject for 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days of a 28-daycycle; (iii) administering 5-azacytidine daily to the subject for 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21days of a 28-day cycle; and (iv) repeating steps (i) to (iii) after 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or21 days of a resting period.

In one embodiment provided herein, the pharmaceutical composition of5-azacytidine includes about 50 mg, about 75 mg, about 100 mg, about 100mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 360mg, about 370 mg, about 400 mg, about 470 mg, about 480 mg, about 490mg, about 500 mg, about 550 mg, or about 600 mg of 5-azacytidine.

In one embodiment provided herein, the at least one additionaltherapeutic agent is administered parenterally.

In one embodiment provided herein, the at least one additionaltherapeutic agent is administered orally.

In one embodiment provided herein, the subject is a human.

Incorporation By Reference: All disclosures (e.g., patents,publications, and web pages) referenced throughout this specificationare incorporated by reference in their entireties. In addition, thefollowing disclosures are also incorporated by reference herein in theirentireties: (1) 2008 ASCO poster abstract by Skikne et al., Leukemia,2008, 22, 1680-84.

III. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. All publications and patents referred to herein areincorporated by reference herein in their entireties.

As used in the specification and the accompanying claims, the indefinitearticles “a” and “an” and the definite article “the” include plural aswell as singular referents, unless the context clearly dictatesotherwise.

The term “about” or “approximately” means an acceptable error for aparticular value as determined by one of ordinary skill in the art,which depends in part on how the value is measured or determined. Incertain embodiments, the term “about” or “approximately” means within 1,2, 3, or 4 standard deviations. In certain embodiments, the term “about”or “approximately” means within 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%,5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, or 0.05% of a given value or range.

As used herein, and unless otherwise specified, the terms “treat,”“treating” and “treatment” refer to the eradication or amelioration of adisease or disorder, or of one or more symptoms associated with thedisease or disorder. In certain embodiments, the terms refer tominimizing the spread or worsening of the disease or disorder resultingfrom the administration of one or more prophylactic or therapeuticagents to a subject with such a disease or disorder. In someembodiments, the terms refer to the administration of a compound ordosage form provided herein, with or without one or more additionalactive agent(s), after the onset of symptoms of the particular disease.

As used herein, amelioration of the symptoms of a particular disorder byadministration of a particular pharmaceutical composition refers to anylessening, whether permanent or temporary, lasting or transient, thatcan be attributed to or associated with administration of thecomposition.

As used herein, and unless otherwise specified, the terms“therapeutically effective amount” and “effective amount” of a compoundmean an amount sufficient to provide a therapeutic benefit in thetreatment or management of a disease or disorder, or to delay orminimize one or more symptoms associated with the disease or disorder. A“therapeutically effective amount” and “effective amount” of a compoundmean an amount of therapeutic agent, alone or in combination with one ormore other agent(s), which provides a therapeutic benefit in thetreatment or management of the disease or disorder. The terms“therapeutically effective amount” and “effective amount” can encompassan amount that improves overall therapy, reduces or avoids symptoms orcauses of disease or disorder, or enhances the therapeutic efficacy ofanother therapeutic agent.

“Tumor,” as used herein, refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all pre-cancerous andcancerous cells and tissues. “Neoplastic,” as used herein, refers to anyform of dysregulated or unregulated cell growth, whether malignant orbenign, resulting in abnormal tissue growth. Thus, “neoplastic cells”include malignant and benign cells having dysregulated or unregulatedcell growth.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include, but are not limitedto blood borne (e.g., lymphoma, leukemia) and solid tumors.

The terms “composition,” “formulation,” and “dosage form,” as usedherein are intended to encompass compositions comprising the specifiedingredient(s) (in the specified amounts, if indicated), as well as anyproduct(s) which result, directly or indirectly, from combination of thespecified ingredient(s) in the specified amount(s). By “pharmaceutical”or “pharmaceutically acceptable” it is meant that any diluent(s),excipient(s) or carrier(s) in the composition, formulation, or dosageform are compatible with the other ingredient(s) and not deleterious tothe recipient thereof. Unless indicated otherwise, the terms“composition,” “formulation,” and “dosage form” are used hereininterchangeably.

The term “immediate release,” when used herein in reference to acomposition, formulation, or dosage form provided herein, means that thecomposition, formulation, or dosage form does not comprise a component(e.g., a coating) that serves to delay the spatial and/or temporalrelease of some or all of the API from the composition, formulation, ordosage form beyond the stomach following oral administration. In certainembodiments, an immediate release composition, formulation, or dosageform is one that releases the API substantially in the stomach followingoral administration. In specific embodiments, an immediate releasecomposition, formulation, or dosage form is one that is notdelayed-release. In specific embodiments, an immediate releasecomposition, formulation, or dosage form is one that does not comprisean enteric coating.

The term “non-enteric-coated,” when used herein, refers to apharmaceutical composition, formulation, or dosage form that does notcomprise a coating intended to release the active ingredient(s) beyondthe stomach (e.g., in the intestine). In certain embodiments, anon-enteric-coated composition, formulation, or dosage form is designedto release the active ingredient(s) substantially in the stomach.

The term “substantially in the stomach,” when used herein in referenceto a composition, formulation, or dosage form provided herein, meansthat at least about 99%, at least about 95%, at least about 90%, atleast about 85%, at least about 80%, at least about 75%, at least about70%, at least about 65%, at least about 60%, at least about 55%, atleast about 50%, at least about 45%, at least about 40%, at least about35%, at least about 30%, at least about 25%, at least about 20%, atleast about 15%, or at least about 10% of 5-azacytidine is released inthe stomach. The term “released in the stomach” and related terms asused herein refer to the process whereby 5-azacytidine is made availablefor uptake by or transport across cells lining the stomach and then madeavailable to the body.

The term “subject” is defined herein to include animals such as mammals,including, but not limited to, primates (e.g., humans), cows, sheep,goats, horses, dogs, cats, rabbits, rats, mice and the like. In specificembodiments, the subject is human.

EXAMPLES Example 1 Materials and Methods:

Cells, Culture Conditions and Reagents

AML cell lines were purchased from the American Tissue CultureCollection (ATCC) or were obtained from the Celgene master cell linebank. Cells were cultured in RPMI 1640 medium supplemented with 10% or20% fetal bovine serum and 10 mM L-glutamine at 37° C. in a humidifiedatmosphere with 5% CO₂. BaF3-FLT3 wt, BaF3-FLT3ITD, BaF3-FLT3D538Y weregenerated by Kyinno (Beijing, China). Cells were grown in RPMI with 10%FBS with 0.5 ug/ml puromycin. Exponentially growing cells were used forall in vitro studies. 5-azacytidine (10 mM in DMSO) was obtained fromthe Celgene compound collection bank was obtained from the Celgenecompound collection bank. Gilteritinib (ASP2215), Midostaurin (PKC412),venetoclax (ABT-199), quizartinib (AC220), the pan caspase inhibitorZ-VAD-FMK were purchased from Selleckchem (Houston, Tex.) andreconstituted as a 10 mM stock in DMSO.

Cell Viability Assay

Cells were plated in 384-well plates (Coming Cat #3764) at 2000cells/well in 50 μl medium. Relative cell numbers, calculated as % DMSOcontrol well, were measured using Cell Titer-Glow (Promega, Madison,Wis.)) according to the manufacturer's instructions. Luminescence valueswere quantified at the time indicated using an EnvVsion plate reader(PerkinElmer). Cells were treated daily with 5-azacytidine for threedays and/or once with midostaurin, gilteritinib or venetoclax. Ninedoses of 5-azacytidine titrated depending on sensitivity to5-azacytidine were combined with six doses of the second drug evaluated,yielding 54 possible combinations, each evaluated in duplicate for everyexperiment. Prism version 7.03 (Prism Software Corporation) was used tocalculate EC₅₀ values.

Data Analysis of Combination Effects

Cell survival was plotted as a function of drug concentration and usedto calculate EC₅₀ values using GraphPad Prism software (San Diego,Calif.). Synergy indices were calculated by the highest single agentmodel and Combenefit software) Combenefit: an interactive platform forthe analysis and visualization of drug combinations (Di Veroli et al.,Bioinformatics. 2016 Sep. 15; 32(18):2866-8).

Western Blots

After treatment with 5-azacytidine and/or inhibitors at the indicateddoses/times, protein was harvested using lysis buffer (Cell Signaling,#9803, Cell Signaling Technologies, Danvers, Mass.) containing 1 mMPMSF. Lysates were quantified using a bicinchoninic acid (BCA) kit(Piece/Thermo Fisher, Waltham, Mass.). 20 to 30 μg protein was resolvedon a 4-12% SDS-PAGE gel, transferred to PVDF membranes (80V/90 minute bywet-transfer), and blocked with Oddysey TBS blocking buffer for 1 hourand then probed with appropriate primary antibodies overnight at 4° C.using dilution as recommended by manufacturer. Membranes were washedthree times for a total of 30 minutes and then incubated with secondaryantibodies at room temperature in the dark for 1 hour. After anotherthree washes, Odyssey infrared imaging system and companion software(LI-COR biosciences, Lincoln, Nebr., USA) were used to scan immunoblotmembranes and to quantify band intensity according to the manufacturer'sinstructions. The ratio of proteins of interest to loading control intreated samples was normalized to the corresponding ratio in untreatedcells. Antibodies used for immunoblotting were purchased from thefollowing sources: BCL-2 (sc-7382), MCL1-(sc-819) from Santa CruzBiotechnology (Dallas, Tex., USA, Bim (2819), caspase-3 (9664) from CellSignaling Technology; beta-Actin (A2228) from Sigma-Aldrich; DNMTI(ab188453) from AbCam; IRDye 680 goat anti-rabbit and IRDye 800 goatanti-mouse secondary antibodies (#925-68073 and #925-32212) werepurchased from Li-COR Biosciences (Lincoln, Nebr.).

RNASeq Analysis

MV4-11 cells were treated with PBS or 1 μM of 5-azacytidine for 24 hoursor daily with 1 μM of 5-azacytidine for 48 hours in triplicate. Aftertreatment, cells were recovered, washed once in PBS, and flash frozen ascell pellets. Cell pellets were sent to Canopy Biosciences for RNAextraction and library preparation and sequencing. RNA was extractedusing the Qiagen RNeasy Mini Kit according to manufacturer'sinstructions. A modified protocol was used to preserve miRNA species.Total RNA Seq libraries were prepared using 200 ng of total RNA and theNEBNext Ultra II Directional Library prep kit. rRNA depletion wasperformed using an RNase-H based method (New England Biolabs, Ipswich,Mass.). MCL1 RNA levels in other cell lines were quantified by RNASequsing standard methods.

Libraries were multiplexed and sequenced using Illumina HiSeq. All genecounts were then imported into the R/Bioconductor package EdgeR and TMMnormalization size factors were calculated to adjust for samples fordifferences in library size. Ribosomal genes and genes not expressed inthe smallest group size minus one samples greater than onecount-per-million were excluded from further analysis. Differentialexpression analysis was then performed to analyze for differencesbetween conditions and the results were filtered for only those geneswith Benjamini-Hochberg false-discovery rate adjusted p-values less thanor equal to 0.05. Global perturbations in known Gene Ontology (GO) termsand KEGG pathways were detected using the R/Bioconductor package GAGE totest for changes in expression of the reported log 2 fold-changesreported by Limma in each term versus the background log 2 fold-changesof all genes found outside the respective term. The R/Bioconductorpackage heatmap and Pathview was used to display heatmaps or annotatedKEGG graphs across groups of samples for each GO term or KEGG pathway(respectively) with a Benjamini-Hochberg false-discovery rate adjustedp-value less than or equal to 0.05. To find differentially expressedgenes, the raw counts were variance stabilized with the R/Bioconductorpackage DESeq2.

To validate ATF3 and SCD expression, MV4-11 cells were treated with PBSor 0.3 μM 5-azacytidine, 1 μM 5-azacytidine, or 3 μM 5-azacytidine for24 hours and 48 hours. At this time, cells were recovered and RNA wasextracted using Qiagen RNeasy kit according to manufacturer'sinstructions. Reverse transcription was performed using SuperScript VILOcDNA synthesis kit. Validated Taqman probes and Taqman Fast AdvancedMaster Mix was used with Viia 7 Real-Time PCR System(Invitrogen/ThermoFisher Scientific, Waltham, Mass.) to quantifytranscripts of ATF3, SCD, and 18S mRNA.

Interfering RNA Gene Silencing

ATF3, SCD, or control Silencer Select siRNAs (16 nM siRNA, Invitrogen)were transfected into MV4-11 cells using Lipofectamine 2000 according tothe manufacturer's suggested protocol. Untreated cells were mocktransfected without siRNA. Cells were then treated with varyingconcentrations of 5-azacytidine daily for 3 days. At day 4, cells weretreated with venetoclax, followed by examination of cell viability atday 7 using Cell Titer Glo according to manufacturer's protocol. Synergywas calculated using Combenefit and compared using Highest Single Agentanalysis.

Confirmation of gene knockdown was performed on siRNA transfected cellsat 72 hours after transfection (without 5-azacytidine or venetoclaxtreatment). RNA was extracted using Qiagen RNeasy kit, and reversetranscription was performed using SuperScript VILO cDNA synthesis kit.Validated Taqman probes and Taqman Fast Advanced Master Mix was usedwith Viia 7 Real-Time PCR System (Invitrogen/ThermoFisher Scientific,Waltham, Mass.) to quantify transcripts of ATF3, SCD, and 18S mRNA.

In Vivo Analysis of 5-Azacytidine Combinations with LEED5-Azacytidine—and HELD 5-Azacytidine Dosing

As used throughout the Examples, LEED refers to the delivery of5-azacytidine at a low exposure for an extended duration (LEED) at 1mg/kg, once daily for fifteen days (QD×15). To deliver the samecumulative dose of 5-azacytidine, the 5-azacytidine is administered at ahigh exposure for a limited duration (HELD), at 3 mg/kg, once daily forfive days (QD×5). LEED models oral administration of AZA, while HELDmodels intravenous or subcutaneous administration of AZA.

Experiments were carried out at Charles River Laboratories (Morissville,N.C.) with female NOD/SCID mice (NOD.CB17-Prkdcscid/NcrCrl, CharlesRiver) that were eight weeks old with a body weight (BW) range of 17.6to 28.4 grams on Day 1 of the study. The animals were fed ad libitumwater (reverse osmosis, 1 ppm Cl), and NIH 31 Modified and IrradiatedLab Diet® consisting of 18.0% crude protein, 5.0% crude fat, and 5.0%crude fiber. The mice were housed on irradiated Enrich-o'Cobs™Laboratory Animal Bedding in static microisolators on a 12-hour lightcycle at 20-22° C. (68-72° F.) and 40-60% humidity.

Celgene provided LEED 5-azacytidine (Lot No. SOOL10), HELD 5-azacytidine(Lot Nos. SOOL10 and SOOL13), midostaurin (MedChemExpress, MonmouthJunction, N.J.), gilteritinib (Sigma Aldrich, St. Louis, Mo.), andvenetoclax (ABT-199, Sigma Aldrich, St. Louis, Mo.). The vehicle used inthis study was 6% Gelucire® 44/14 (Gattefossé, Paramus, N.J.) indeionized (DI) water, which was a waxy solid that required a water bathheat to 44° C. for melting, dosed PO (per oral), and phosphate bufferedsaline (PBS), dosed IP (intraperitoneal). On each day of dosing, anappropriate amount of LEED or HELD 5-azacytidine was resuspended in PBSto yield a dosing suspension at 0.1 mg/mL or 0.3 mg/mL, respectively. Oneach day of dosing, an appropriate amount of midostaurin was dissolvedin 6% Gelucire 44/14 to yield a dosing solution at 10 mg/mL. On each dayof dosing, an appropriate amount of gilteritinib was dissolved in 0.5%methylcellulose in DI water to yield a dosing solution at 0.4 mg/mL.Each week, an appropriate amount of venetoclax was dissolved in 10%ethanol:30% PEG400:60% phosal 50 propylene glycol to yield a dosingsolution at 10 mg/mL. Cells used for inoculation were harvested duringlog phase growth and resuspended at a concentration of 5×107 cells/mL inPBS. Each test mouse received 5×10⁶ MOLM-13 cells or 10⁷ MV4-11 cells(0.2 mL cell suspension) by tail vein injection. Dosing was initiatedthree days after tumor cell inoculation, which was designated as Day 1of the study. NOD/SCID mice (n=9-12/group) were randomized according tobody weight and dosed. Phosphate buffered saline, LEED 5-azacytidine,and HELD 5-azacytidine were administered intraperitoneally (IP), whilemidostaurin, gilteritinib, and venetoclax were administered PO. Vehiclewas administered both IP and PO. The dosing volume for all treatmentswas 10 mL/kg, scaled to the weight of each individual animal.

Animals were monitored individually for an endpoint of moribundity dueto progression of the leukemia. Full hind limb paralysis, severe ocularproptosis, or moribundity was considered sufficient for euthanasia dueto tumor progression. Moribund animals were defined as sick animalsunable to reach food and water. These deaths were classified as death onsurvival study. The time to endpoint (TTE), in days, was recorded foreach mouse that died of its disease or was euthanized due to extensivetumor progression. Animals that did not reach the endpoint wereeuthanized at the end of the study and were assigned a TTE value equalto the last day. An animal classified as having died fromtreatment-related (TR) causes was assigned a TTE value equal to the dayof death. An animal classified as having died from non-treatment-related(NTR) causes, or used for sampling before endpoint, was excluded fromTTE calculations and all further analyses. The median TTE value wascalculated for each group. The median TTE of treated mice was expressedas a percentage of the median TTE of the control mice (% T/C), and theincrease in life span (ILS) was calculated as: ILS=% T/C−100%, whereT=median TTE treated, and C=median TTE control. Thus, if T=C, ILS=0%.

Animals were weighed daily on Days 1-5, then twice per week until thecompletion of the study. The mice were observed frequently for overtsigns of any adverse, treatment-related (TR) side effects, and clinicalsigns were recorded when observed. Individual body weight loss wasmonitored as per protocol and any animal that exceeded the limits foracceptable body weight loss was euthanized. Group mean body weight lossalso was monitored as per protocol. Dosing was suspended in any groupthat exceeded the limits for acceptable mean body weight loss. If meanbody weight recovered, then dosing may be resumed in that group, but ata lower dosage or less frequent dosing schedule. Acceptable toxicity forthe maximum tolerated dose was defined as a group mean body-weight lossof less than 20% during the study and not more than one TR death amongten treated animals. A death was classified as TR if attributable totreatment side effects as evidenced by clinical signs and/or necropsy ormay also be classified as TR if due to unknown causes during the dosingperiod or within 14 days of the last dose. A death was classified as NTRif there was no evidence that death was related to treatment sideeffects or tumor progression. Non-treatment-related deaths may befurther characterized based on cause of death. A death may be classifiedas NTRa if it resulted from an accident or human error. A death may beclassified as NTRu if the cause of death is unknown and there is noavailable evidence of death related to treatment side effects,metastasis, accident or human error, although death due to theseetiologies cannot be excluded. Survival was analyzed by the Kaplan-Meiermethod, based on TTE values. The logrank (Mantel-Cox) andGehan-Breslow-Wilcoxon tests determined the significance of thedifference between the overall survival experiences (survival curves) oftwo groups, based on TTE values.

Results

Combinations of 5-Azacytidine with Midostaurin, Gilteritinib orVenetoclax

FIGS. 1-7 provide various information and data regarding the experiment.For example, FIG. 1 is a bar graph representing the maximum EC₅₀ foldshift of 5-azacytidine in combination with gilteritinib, and5-azacytidine in combination with midostaurin, both with cell linesMV4-11 and MOLM-13. The results from three different dosing schedulesare shown: (i) 5-azacytidine administered first (black bar); (ii) thetwo agents administered concurrently (light gray bar); and (iii)5-azacytidine administered second (medium gray bar). FIG. 2 representsthe three different dosing schedules of (i) 5-azacytidine (AZA)administered first at intervals before the FLT3 inhibitor (FLT3i); (ii)the two agents (5-azacytidine and FLT3i) administered concurrently; and(iii) 5-azacytidine administered second at intervals after the FLT3i isadministered; where the FLT3i may be any suitable FLT3 inhibitor, suchas midostaurin or gilteritinib. FIGS. 3A-D represent the maximum EC₅₀fold shift of 5-azacytidine in combination with venetoclax with celllines MV4-11 (FIG. 3A) and MOLM-13 (FIG. 3C). Three different dosingschedules are shown, (i) 5-azacytidine administered first (black bar);(ii) the two agents administered concurrently (light gray bar); and(iii) 5-azacytidine administered second (medium gray bar). A synergyindex is also shown for 5-azacytidine administered in combination withvenetoclax with cell lines MV4-11 (FIG. 3B) and MOLM-13 (FIG. 3D) forthe three different dosing schedules.

FIGS. 4A-C represent Response Surface Analyses showing synergy of5-azacytidine with venetoclax in MV4-11 cells when 5-azacytidine isadministered first (FIG. 4A), the relatively lower synergy withsimultaneous administration (FIG. 4B), and synergy with venetoclaxadministered first (FIG. 4C). Response surface methodology (RSM)explores the statistical relationships between several explanatoryvariables and one or more response variables. RSM uses a sequence ofdesigned experiments to obtain an optimal response, which in the presentcase is the synergistic effects of 5-azacytidine with venetoclax.

FIG. 5 depicts a western blot showing that (a) 5-azacytidine andmidostaurin (“aza+0.3 μM Mido”) and (b) 5-azacytidine and gilteritinib(“aza+0.3 μM Gilt”) augment MCL-1 degradation in MV4-11 cell lines. Inaddition, FIG. 6 depicts a western blot showing that 5-azacytidine andvenetoclax treatment decreases MCL-1 Levels in FLT3ITD MV4-11 cells.

Finally, FIGS. 7A-C depict in vivo assessments of 5-azacytidinecombinations in a MOLM-13 xenograft model, with a graph of percentsurvival (y-axis) vs day 0 to 70 (x-axis). Dosing for the experimentsshown in FIGS. 7A-C was as follows: (i) 5-azacytidine (low exposure,extended duration, LEED): 1 mg/kg interperitoneally (IP), once daily forfive days, three times (qd×5×3); (ii) 5-azacytidine (high exposure,limited duration, HELD): 3 mg/kg interperitoneally (IP), once daily forfive days (qd×5); (iii) Midaustaurin (100 mg/kg orally (PO), once dailyfor twenty-one days (qd×21)); (iv) Gilteritinib (4 mg/kg orally (PO),once daily for twenty-one days (qd×21)); and (v) Venetoclax (100 mg/kgorally (PO), once daily for twenty-one days (qd×21)). P-value (relativeto best single agent) *P<0.05; **P<0.001; ***P<0.0001. FIG. 7A shows theresults of the combination of 5-azacytidine and midostaurin, FIG. 7Bshows the results of 5-azacytidine combined with venetoclax, and FIG. 7Cshows the results of the combination of 5-azacytidine and gilteritinib.For FIG. 7A, the compositions tested were vehicle, 5-azacytidine (lowexposure, extended duration, LEED, schedule of 1 mg/kg 5-azacytidine,once daily for fifteen days (qd×15)), 5-azacytidine (high exposure,limited duration, HELD, schedule of 3 mg/kg 5-azacytidine, once dailyfor five days (qd×5)), midostaurin (100/kg, once daily for twenty eightdays (qd×28)), LEED+midostaurin, and HELD+midostaurin. For FIG. 7B, thecompositions tested were vehicle, 5-azacytidine (LEED), 5-azacytidine(HELD), venetoclax, LEED+venetoclax, and HELD+venetoclax. For FIG. 7C,the compositions tested were vehicle, 5-azacytidine (LEED),5-azacytidine (HELD), gilteritinib, LEED+gilteritinib, andHELD+gilteritinib. Both LEED and HELD 5-azacytidine dosing causedstatistically significantly increases in survival compared to vehiclealone (LEED vs vehicle, p=0.003 by Gehan-Breslow-Wilcoxon test; HELD vsvehicle, p=0.003 by Gehan-Breslow-Wilcoxon test). Midostaurin alone andin combination with LEED or HELD 5-azacytidine significantly increasedsurvival compared to vehicle alone (Midostaurin vs vehicle, p=0.027;LEED+midostaurin vs vehicle, p=0.012; HELD+midostaurin vs vehicle,p=0.003). HELD 5-azacytidine dosing in combination with midostaurinsignificantly increased survival compared to LEED or HELD 5-azacytidine,respectively (LEED+midostaurin vs LEED, p=0.028; HELD+midostaurin vsHELD, p=0.039). No significant changes in survival were observed betweenLEED or HELD in combination with midostaurin compared to midostaurintreatment alone. Median survival was increased with LEED or HELD5-azacytidine in combination with midostaurin compared to vehicle orsingle agents (LEED+midostaurin=45 days, HELD+midostaurin=43 days,vehicle=19 days, midostaurin=34 days, LEED=36 days, HELD=32 days, (FIG.7A). Gilteritinib alone and in combination with LEED or HELD5-azacytidine significantly increased survival compared to vehicle alone(gilteritinib vs vehicle, p=0.003; LEED+gilteritinib vs vehicle,p=0.003; HELD+gilteritinib vs vehicle, p=0.003). Low exposure, extendedduration or HELD 5-azacytidine dosing in combination with gilteritinibsignificantly increased survival compared to either LEED or HELD5-azacytidine alone (LEED+gilteritinib vs LEED, p=0.019;LEED+gilteritinib vs HELD, p=0.004; HELD+gilteritinib vs LEED, p=0.008;HELD+gilteritinib vs HELD, p=0.003. Furthermore, LEED or HELD5-azacytidine dosing in combination with gilteritinib significantlyincreased survival compared to gilteritinib alone (LEED+gilteritinib vsgilteritinib, p<0.001; HELD+gilteritinib vs gilteritinib, p<0.001).Venetoclax alone and in combination with LEED or HELD 5-azacytidinesignificantly increased survival compared to vehicle alone (venetoclaxvs vehicle, p=0.003; LEED+venetoclax vs vehicle, p=0.002;HELD+venetoclax vs vehicle, p=0.004) (FIG. 7B). Low exposure, extendedduration or HELD 5-azacytidine dosing in combination with venetoclaxsignificantly increased survival compared to either LEED or HELD5-azacytidine alone (LEED+venetoclax vs LEED, p=0.001; LEED+venetoclaxvs HELD, p<0.001; HELD+venetoclax vs LEED, p=<0.001; HELD+venetoclax vsHELD, p=<0.001. Furthermore, LEED or HELD 5-azacytidine dosing incombination with venetoclax significantly increased survival compared tovenetoclax alone (LEED+venetoclax vs venetoclax, p<0.001;HELD+venetoclax vs venetoclax, p<0.001). Low exposure, extended durationin combination with venetoclax was not significantly different than HELDin combination with venetoclax. Median survival was increased with LEEDor HELD 5-azacytidine in combination with venetoclax compared to vehicleor single agents (LEED+venetoclax=46 days, HELD+venetoclax=45 days,vehicle=19 days, venetoclax=29 days, LEED=36 days, HELD=32 days). Mediansurvival was increased with LEED or HELD 5-azacytidine in combinationwith gilteritinib compared to vehicle or single agents(LEED+gilteritinib=45 days, HELD+gilteritinib=43 days, vehicle=19 days,gilteritinib=34 days, LEED=36 days, HELD=32 days, (FIG. 7C).

FIGS. 8A, 8B, and 8C show the sensitivity of 22 AML cell lines to5-azacytidine (AZA) and venetoclax as single agents (FIGS. 8A and 8B,respectively) and the combination of 5-azacytidine and venetoclax (FIG.8C). FIG. 8A shows that 5-azacytidine showed cytotoxic effects in mostcell lines, with EC₅₀ values ranging from 0.15 μM to 2.5 μM. Incontrast, FIG. 8B shows that 11/22 of the AML cell lines examined weresensitive to venetoclax (EC₅₀<10 μM). FIG. 8C shows the combinatorialactivity of 5-azacytidine with venetoclax using surface responseanalysis and highest single agent model, where 10/22 cell lines showedsynergistic activity above the arbitrary threshold of 20. Notably, threecell lines that were resistant to venetoclax (Kasumi-1, Kasumi-2 andNOMO-1) showed reversal of venetoclax resistance with co-treatment with5-azacytidine. Cell lines that carried FLT3-ITD, a recurrent mutation inAML, also showed synergistic activity with 5-azacytidine and venetoclax.

These results surprisingly demonstrate that the combination of5-azacytidine with venetoclax provides a synergistic effect in AML celllines, and in particular AML cell lines that are resistant tovenetoclax. The results support that the combination of 5-azacytidinewith venetoclax is safe and effective for treating AML patients.

Whether the specific schedule of 5-azacytidine and venetoclaxadministration has an influence on the synergistic effect provided by5-azacytidine and venetoclax was also investigated. FIGS. 9A-F show thecell survival of MV4-11 cells (FIGS. 9A-C) and MOLM-13 cells (FIGS.9D-F) seven days after the start of treatment with 5-azacytidine andvenetoclax. The following schedules were tested: 5-azacytidineadministration on Days 1, 2 and 3, followed by venetoclax administrationon Day 4 (5-azacytidine (AZA) First) (FIGS. 9A and 9D); 5-azacytidineand venetoclax co-administration on Day 1, followed by 5-azacytidineadministration on Days 2 and 3 (Simultaneous) (FIGS. 9B and 9E); andvenetoclax administration on Day 1, followed by 5-azacytidine on Days 2,3 and 4 (venetoclax first) (FIGS. 9C and 9F). The results show that forboth cell lines, the regimen where 5-azacytidine was administered firstprovided the maximal synergistic effects. These results suggest that5-azacytidine that may prime AML cells for venetoclax activity.

One of the factors for venetoclax resistance is the expression of theapoptotic regulator MCL-1, which is upregulated in FLT3 mutated AML andis downregulated after 5-azacytidine treatment. To examine whether MCL-1levels correlate with the degree of the synergistic effect of thecombination of 5-azacytidine and venetoclax, a panel of engineered BaF3cell lines expressing either wild-type FLT3, FLT3-ITD or FLT3 (D835Y)mutations was examined. Engineered BaF3 cell lines also proliferatedindependently of IL-3. FIG. 10A shows that these engineered BaF3 celllines were resistant to venetoclax (EC₅₀>1 μM), but sensitive to FLT3inhibitors, such as gilteritinib, midostaurin and quizartinib. The datashown in FIG. 10A is also shown in Table 1, below.

TABLE 1 BaF3 cells expressing various FLT3 (EC50, nM) BaF-FLT3(WT)BaF-FLT3-ITD BaF-FLT3(D835Y) AZA 189 598 456 Gilteritinib 4 1.8 1.8Midostaurin 23 5.5 5.4 Quizartinib 3 0.008 9.5 Venetoclax >10000 >100004332

FIG. 10B shows that MCL-1 was detected in all lines, with the highestexpression levels observed in the FLT-ITD mutant line, followed by FLT3(D835Y). The combination of 5-azacytidine with venetoclax showed asynergistic effect, with the highest synergy index observed in FLT3(wildtype), expressing the lowest levels of MCL-1, followed by FLT3(D835Y) (intermediate MCL-1 levels) and FLT3-ITD (highest MCL-1) (FIG.10C). These results suggests that MCL-1 expression may be a determinantfactor for the 5-azacytidine-venetoclax synergy.

To further explore the relationship between MCL-1 and5-azacytidine-venetoclax synergy further, the relationship between MCL1RNA levels and 5-azacytidine-venetoclax synergy indices was examinedexplored in a panel of 20 AML cell lines. FIG. 11 shows that MCL1 RNAlevels correlated directly with the synergy index (r²=−0.5607, p=0.0101)in a panel of 20 AML cell lines. These results show that MCL-1 may be akey regulator for AZA priming for venetoclax-induced apoptosis,specifically 5-azacytidine may lower MCL-1 below a certain threshold toallow venetoclax-mediated apoptosis.

Next, the extent of 5-azacytidine-mediated MCL-1 degradation in fourdifferent AML cell lines was explored KG1a (FIG. 12A), MV4-11 (FIG.12B), THP-1 (FIG. 12C) and OCI-AML-2 (FIG. 12D). The results showed5-azacytidine-venetoclax synergistic activity with KG1a (FIG. 12E) andMV4-11 (FIG. 12F) cell lines (synergy index (SI) of 70 and 35.5,respectively) and very little or no synergistic activity with THP-1(FIG. 12G) and OCI-AML-2 (FIG. 12H) cell lines (SI of 20.2 and 10.8,respectively). For the KG1a (FIG. 12A) and MV4-11 (FIG. 12B) cell lines,where 5-azacytidine-venetoclax had the greatest synergistic effect(FIGS. 12E and 12F), 5-azacytidine led to MCL-1 degradation the fastest,starting 6 hours after treatment. In contrast, for THP-1 (FIG. 12C),where 5-azacytidine-venetoclax only provided minor synergistic activityshowed 5-azacytidine-mediated MCL-1 degradation later, starting at 16hours, with incomplete degradation by 24 hours (FIG. 12G). For OCI-AML2(FIG. 12D), where 5-azacytidine-venetoclax had the lowest synergisticeffect (FIG. 12H), 5-azacytidine treatment did not lead to anydegradation of MCL-1. These results support the hypothesis that5-azacytidine primes cells for venetoclax-mediated apoptosis by loweringMCL-1 levels.

One possible mechanism by which 5-azacytidine downregulates MCL-1 is byinducing caspase activation. Caspase activation can be assayed byevaluating the degradation of caspase 3 in a western blot (FIG. 13A). Tofind out whether this effect is caspase-dependent, the cells weretreated with Z-VAD-FMK, a pan-caspase inhibitor, and the extent of MCL-1degradation by 5-azacytidine was measured (FIG. 13B). In particular,FIG. 13B shows a bar graph of MCL-1 degradation by 5-azacytidine, wherecells were treated with 20 μM Z-VAD-FMK for 1 hours before 5-azacytidinetreatment for another 16 hours. Caspase inhibition partially ablatedMCL-1 degradation by 5-azacytidine in MV4-11 cells, suggestingadditional, caspase-independent mechanisms of MCL-1 degradation. It wasfound that Z-VAD-FMK partially ablated the ability of 5-azacytidine todegrade MCL-1, suggesting this process is mediated by caspase-dependentand independent mechanisms.

To further understand how 5-azacytidine primes venetoclax for acuteapoptosis, RNAseq was performed on MV4-11 cells treated with PBS(vehicle), 1 μM AZA for 24 hours (FIG. 14A), or with 1 μM AZA for 48hours (FIG. 14B). Table 2 is the pathway analysis for RNASeq data inFIG. 14A and shows the analysis after 5-azacytidine treatment, which wasthe categorization of genes that were significantly induced or repressedby 5-azacytidine based on KEGG pathways.

TABLE 2 Significant Kegg pathways at 24 h (none significant at 48 h)Adjusted p Pathway Mean logFC p-value value Ribosomes −10.50 10.495475.44E−21 Oxidative phosphorylation −7.32 7.315598 2.01E−12 Metabolicpathways −5.29 5.788892 4.06E−09 Cell cycle −5.75 5.746475 1.43E−08Protein processing in −4.96 4.958095 6.26E−07 endoplasmic reticulum DNAreplication −4.45 4.450573 1.83E−05 Proteosome −4.41 4.410196 2.17E−05Phagosome −4.13 4.134259 2.49E−05 Spliceosome −4.10 4.100521 2.90E−05Necroptosis −3.78 3.779974 9.92E−05 Fatty acid metabolism −3.43 3.4252494.51E−04 Antigen processing and −3.39 3.393502 5.02E−04 presentationCarbon metabolism −3.35 3.354515 4.75E−04 Biosynthesis of −3.26 3.2605861.11E−03 unsaturated fatty acids Protein export −3.19 3.191372 1.46E−03RNA transport −3.08 3.081532 1.15E−03 Mismatch repair −2.98 2.9756212.43E−03 Fatty acid elongation −2.87 2.867612 3.26E−03 Lysosome −2.822.816478 2.65E−03 Cellular senescence −2.69 2.691944 3.78E−03 Cardiacmuscle contraction −2.61 2.614263 5.21E−03 Terpenoid backbone −2.482.481832 9.05E−03 biosynthesis Regulation of actin −2.46 2.4562627.30E−03 cytoskeleton Taste transduction 2.44 2.436918 8.81E−03 p53signaling pathway −2.42 2.419681 8.46E−03

No significant differences were observed in KEGG pathways regulated by5-azacytidine after 48 hours of treatment. However, the top KEGGpathways differentially regulated after 24 hours of 5-azacytidinetreatment were “Ribosome”, “Oxidative Phosphorylation”, “MetabolicPathways”, and “Cell Cycle”. These results support the hypothesis that5-azacytidine has a role in altering cell metabolism, causing cell cyclearrest, suppressing oxidative phosphorylation, which was previouslyobserved in patients treated with 5-azacytidine+venetoclax combination.

Volcano plots of significantly modified genes at 24 hours (FIG. 14A) and48 hours (FIG. 14B) show 5-azacytidine induced 133 differentiallyexpressed genes at 24 hours and 226 differentially expressed genes at 48hours. Upon further analysis of the 5-azacytidine-induced differentiallyexpressed genes, two genes were identified that have previously beenshown to regulate MCL1 expression: activating transcription factor 3(ATF3) and stearoyl-CoA desaturase (SCD). ATF3 is a stress responsivetranscription factor that was shown to regulate MCL-1, as well as immuneand metabolic genes. ATF3 expression was increased two-fold 48 hoursafter 5-azacytidine treatment. On the other hand, the expression of SCD,a regulator of lipid metabolism and MCL1, was decreased 2.5-fold by5-azacytidine treatment at 48 hours. Alterations in ATF3 (FIG. 14C) andSCD (FIG. 14D) expression were validated in a separate experiment usingreal-time PCR. ATF3 expression was increased in a time- andconcentration-dependent manner, as 0.3 μM 5-azacytidine treatment wasnot sufficient to induce ATF3 expression at either 24 or 48 hours (FIG.14C). Similarly, SCD expression was decreased rapidly within 24 hourswhen treated with 3 μM 5-azacytidine, although it was not affected bylow concentrations of 5-azacytidine at this timepoint (FIG. 14D).

Given their connection with regulating MCL1 expression, it washypothesized that ATF3 and/or SCD may contribute to5-azacytidine-venetoclax synergy. To explore this further, siRNAknockdown of these genes was utilized in MV4-11 cells to assess theirfunction in synergy. MV4-11 cells were left untransfected or transfectedwith ATF3, SCD, or control (scrambled) siRNA. As a control, cells weretransfected with siRNA and collected for RNA and qPCR 72 hours aftertransfection. (FIG. 15A) This confirmed that siRNA knockdown decreased,but did not completely ablate, mRNA expression of ATF3 or SCD when cellswere transfected with ATF3 or SCD siRNA, respectively. Furthermore, incells treated with scramble siRNA, no changes in ATF3 (FIG. 15B) or SCD(FIG. 15C) expression were seen. Following transfection, cells weretreated with various concentrations of 5-azacytidine daily for Days 1-3.At Day 4, cells were dosed with venetoclax, followed by cell viabilitytest using CellTiter-Glo® 7 after treatment initiation.5-Azacytidine-venetoclax synergy was calculated using Combenefit andHighest Single Agent analysis (FIGS. 15D-G). 5-Azacytidine-venetoclaxsynergy was confirmed in cells that were not transfected (SynergyIndex=43) (FIG. 15D), and the synergy was not affected by transfectionitself, as cells transfected with scramble siRNA (FIG. 15E) had asynergy index of 46. When ATF3 was knocked down (FIG. 15F),5-azacytidine-venetoclax had decreased synergy (Synergy Index=19). Onthe other hand, when SCD (FIG. 15G) was knocked down,5-azacytidine-venetoclax had increased synergy (Synergy Index=60). Thisdata suggests that 5-azacytidine-induced increases in ATF3 and decreasesin SCD play at least a partial role in 5-azacytidine-venetoclax synergy.

Whether 5-azacytidine and venetoclax have synergistic activity in vivoat doses and schedules corresponding to injectable 5-azacytidine (HELD)or oral 5-azacytidine (LEED) was next evaluated. MV4-11 (FIG. 16A-C) andMOLM-13 (FIGS. D-F), two cell lines that showed 5-azacytidine-venetoclaxsynergy (FIG. 8C), were used to generate disseminated AML xenograft micein immunodeficient animals. In vitro, venetoclax sensitized both celllines to venetoclax (FIGS. 16A and 16D) and synergized with5-azacytidine (FIGS. 16B and 16E). To model oral 5-azacytidine (LEED)regimes, mice were treated with 1 mg/kg 5-azacytidine for 15 days (lowexposure, extended duration). Alternatively, to use the same cumulativedose but with an injectable 5-azacytidine (HELD) regime, mice weretreated with 3 mg/ml 5-azacytidine for 5 days (high exposure, limitedduration).

For MV4-11 implantation, female NCG mice were injected via tail veinwith 1×10⁷ cells in 0.2 mL cell suspension. Day 1 was designated asfourteen days after implantation. On Day 1, mice were sorted intotreatment groups based on body weight and dosing was initiated asfollows: mice treated with vehicle, high dose 5-azacytidine (HELD, 3mg/kg once daily for five days (qd×5)), low dose 5-azacytidine (LEED, 1mg/kg once daily for five days, three times (qd×5×3)), venetoclax (100mg/kg, once daily for twenty one days (qd×21)), HELD+venetoclax, orLEED+venetoclax. Mice were monitored for body weight loss andmoribundity for up to 56 days after initial treatment to determine whenmice succumbed to tumor burden. Venetoclax alone or in combination withLEED or HELD 5-azacytidine significantly increased survival compared tovehicle alone (venetoclax vs vehicle, p=0.0493; LEED+venetoclax vsvehicle, p=0.0123; HELD+venetoclax vs vehicle, p=0.04). LEED or HELD5-azacytidine in combination with venetoclax significantly increasedsurvival compared to 5-azacytidine alone (LEED+venetoclax vs LEED,p=0.001; HELD+venetoclax vs HELD, p=0.0004). However, onlyLEED+5-azacytidine was significantly better than venetoclax alone(LEED+venetoclax vs venetoclax, p=0.0378). Furthermore, LEED or HELD5-azacytidine combination with venetoclax did increase median survivalcompared to single agents (LEED+venetoclax=38, HELD+venetoclax=37,vehicle=29.5, HELD=35, LEED=35, venetoclax=35.5). (FIG. 16C)

These experiments were repeated with a second FLT3-ITD cell line,MOLM-13. Briefly, 5×10⁶ MOLM-13 cells were injected into 12 NOD/SCI miceper group. Three days after tumor cell inoculation, mice were treatedwith the same dosing regimen as MV4-11 cells. Mice were monitored forbody weight loss and moribundity for up to 70 days after initialtreatment to determine when mice succumbed to disease burden. Venetoclaxalone and in combination with LEED or HELD 5-azacytidine significantlyincreased survival compared to vehicle alone (venetoclax vs vehicle,p=0.003; LEED+venetoclax vs vehicle, p=0.002; HELD+venetoclax vsvehicle, p=0.004). Low exposure, extended duration or HELD 5-azacytidinedosing in combination with venetoclax significantly increased survivalcompared to either LEED or HELD 5-azacytidine alone (LEED+venetoclax vsLEED, p=0.001; LEED+venetoclax vs HELD, p<0.001; HELD+venetoclax vsLEED, p=<0.001; HELD+venetoclax vs HELD, p=<0.001. Furthermore, LEED orHELD 5-azacytidine dosing in combination with venetoclax significantlyincreased survival compared to venetoclax alone (LEED+venetoclax vsvenetoclax, p<0.001; HELD+venetoclax vs venetoclax, p<0.001). Mediansurvival was increased with LEED or HELD 5-azacytidine in combinationwith venetoclax compared to vehicle or single agents (LEED+venetoclax=46days, HELD+venetoclax=45 days, vehicle=19 days, venetoclax=29 days,LEED=36 days, HELD=32 days). (FIG. 16F) Altogether, these results showthat patients with Flt3-ITD mutations may benefit from5-azacytidine+venetoclax combination therapy.

Combinations of 5-Azacytidine with FLT-3 Inhibitors

FLT3 mutations occur in ˜30% of AML, patients and have been associatedwith poor prognosis. The broad-acting FLT-3 inhibitor midostaurin andthe selective FLT3 inhibitor, gilteritinib, have been approved for thetreatment of AML. To investigate whether co-treatment with 5-azacytidineand FLT3 inhibitors have a synergistic effect in AML cells, two FLT3-ITDcell lines, MV4-11 and MOLM-13 cells were treated with5-azacytidine+midostaurin or 5-azacytidine+gilteritinib. Cells weretreated with daily doses of 5-azacytidine on Day 1-3, and then treatedwith a FLT-3 inhibitor (midostaurin or gilteritinib) at Day 4. Cellswere collected on Day 7 and cell viability was assessed byCellTiter-Glo® assay. Midostaurin sensitized MV4-11 to 5-azacytidine(FIG. 17A) and showed synergistic activity with 5-azacytidine (FIG.17B). Similar effects were observed in MV4-11 cells treated with5-azacytidine and gilteritinib (FIGS. 17C and 17D), as well as inMOLM-13 cells treated with 5-azacytidine and midostaurin (FIGS. 17E and17F) or gilteritinib (FIGS. 17G and 17 H).

Next synergistic activity between FLT3 inhibitors and 5-azacytidineadministered using a dose and schedule similar to injectable (highexposure, limited duration, or HELD regimen) or oral (low exposure,extended duration, LEED) was examined. Two disseminated xenograft modelsof AML based on MOLM-13 and MV4-11 cell lines were used. Mice weretreated with 5-azacytidine using a HELD regimen (3 mg/kg, daily for 5days) or LEED (1 mg/kg, once daily for fifteen days (qd×15)). FLT3inhibitors midostaurin at 100 mg/kg daily for 21 days and gilteritinibat 4 mg/kg, qd×21 were administered as single agents or with HELD orLEED 5-azacytidine regimens. In MOLM-13 xenograft models, midostaurinalone and in combination with LEED or HELD 5-azacytidine significantlyincreased survival compared to vehicle alone (midostaurin vs vehicle,p=0.027; LEED+midostaurin vs vehicle, p=0.012; HELD+midostaurin vsvehicle, p=0.003) (FIG. 17I). Low exposure, extended duration or HELD5-azacytidine dosing in combination with midostaurin significantlyincreased survival compared to LEED or HELD 5-azacytidine, respectively(LEED+midostaurin vs LEED, p=0.028; HELD+midostaurin vs HELD, p=0.039).No significant changes in survival were observed between LEED or HELD incombination with midostaurin compared to midostaurin treatment alone.Median survival was increased with LEED or HELD 5-azacytidine incombination with midostaurin compared to vehicle or single agents(LEED+midostaurin=45 days, HELD+midostaurin=43 days, vehicle=19 days,midostaurin=34 days, LEED=36 days, HELD=32 days) (FIG. 17I).

In MV4-11 xenograft models, midostaurin alone and in combination withLEED or HELD 5-azacytidine increased survival compared to vehicle alone(Midostaurin vs vehicle, p=0.0067; LEED+midostaurin vs vehicle,p=0.0084; HELD+midostaurin vs vehicle, p=0.0625). LEED or HELD5-azacytidine dosing in combination with midostaurin significantlyincreased survival compared to either LEED or HELD 5-azacytidine alone(LEED+midostaurin vs LEED, p=<0.0001; HELD+midostaurin vs HELD,p=0.0015). Furthermore, LEED or HELD 5-azacytidine in combination withmidostaurin did not significantly increase survival compared tomidostaurin alone (LEED+midostaurin vs midostaurin, p=0.1704;HELD+midostaurin vs midostaurin, p=0.8308). Median survival wasincreased with LEED or HELD 5-azacytidine in combination withmidostaurin compared to vehicle or single agents HELD or LEED(LEED+midostaurin=64.5, HELD+midostaurin=59.5, vehicle=29.5, LEED=35,HELD=35, midostaurin=57) (FIG. 17J).

In MOLM-13 xenograft models, gilteritinib alone and in combination withLEED or HELD 5-azacytidine significantly increased survival compared tovehicle alone (gilteritinib vs vehicle, p=0.003; LEED+gilteritinib vsvehicle, p=0.003; HELD+gilteritinib vs vehicle, p=0.003). Low exposure,extended duration or HELD 5-azacytidine dosing in combination withgilteritinib significantly increased survival compared to either LEED orHELD 5-azacytidine alone (LEED+gilteritinib vs LEED, p=0.019;LEED+gilteritinib vs HELD, p=0.004; HELD+gilteritinib vs LEED, p=0.008;HELD+gilteritinib vs HELD, p=0.003. Furthermore, LEED or HELD5-azacytidine dosing in combination with gilteritinib significantlyincreased survival compared to gilteritinib alone (LEED+gilteritinib vsgilteritinib, p<0.001; HELD+gilteritinib vs gilteritinib, p<0.001).Median survival was increased with LEED or HELD 5-azacytidine incombination with gilteritinib compared to vehicle or single agents(LEED+gilteritinib=45 days, HELD+gilteritinib=43 days, vehicle=19 days,gilteritinib=34 days, LEED=36 days, HELD=32 days) (FIG. 17K).

Altogether, these results suggest that LEED or HELD 5-azacytidine incombination with a FLT3 inhibitor is significantly more effective atkilling AML, cells as compared to single agent 5-azacytidine or FLT3inhibitor alone.

Example 2

Study Objectives: The primary objective of the study is as follows.

Combination Arm A (oral 5-azacytidine+Ivosidenib arm): Objectives are:(1) To establish a maximum tolerated dose (MTD) or a maximumadministered dose (MAD) of oral 5-azacytidine when given in combinationwith Ivosidenib in AML patients with an IDH1 mutation. (2) To establishthe safety and tolerability of oral 5-azacytidine when given incombination with Ivosidenib in AML patients with an IDH1 mutation.

Combination Arm B (oral 5-azacytidine+Enasidenib arm): Objectives are:(1) To establish a maximum tolerated dose (MTD) or a maximumadministered dose (MAD) of oral 5-azacytidine when given in combinationwith Enasidenib in AML patients with an IDH2 mutation. (2) To establishthe safety and tolerability of oral 5-azacytidine when given incombination with Enasidenib in AML patients with an IDH2 mutation.

Combination Arm C (oral 5-azacytidine+FLT3 arm): Objectives are: (1) Toestablish a maximum tolerated dose (MTD) or a maximum administered dose(MAD) of oral 5-azacytidine when given in combination with a FLT3inhibitor in AML patients with a FLT3 ITD or TKD mutation. (2) Toestablish the safety and tolerability of oral 5-azacytidine when givenin combination with a FLT3 inhibitor in AML patients with a FLT3 ITD orTKD mutation.

Combination Arm D (oral 5-azacytidine+venetoclax arm): Objectives are:(1) To establish a maximum tolerated dose (MTD) or a maximumadministered dose (MAD) of oral 5-azacytidine when given in combinationwith venetoclax in AML patients. (2) To establish the safety andtolerability of oral 5-azacytidine when given in combination withvenetoclax in AML patients.

The secondary objectives are as follows: (1) To characterize thepharmacokinetics (PK) of oral 5-azacytidine in combination withIvosidenib, Enasidenib, venetoclax, and a FLT3 inhibitor in AMLpatients; and (2) To assess the preliminary efficacy of oral5-azacytidine in combination with Ivosidenib, Enasidenib, venetoclax,and a FLT3 inhibitor in AML patients.

The exploratory objectives are as follows: (1) To explore therelationships of drug exposure with efficacy, safety, pharmacodynamicsand/or other exploratory endpoints; of oral 5-azacytidine whenadministered with Ivosidenib, Enasidenib, venetoclax, or a FLT3inhibitor. (2) To evaluate the minimal residual disease (MRD), by flowcytometry (or relevant gene sequencing methods (for example, FLT3,IDH1/2 variant allele frequency (VAF), in blood and/or bone marrow. (3)To investigate the pertinent pharmacodynamics markers of oral5-azacytidine and each of the combination partner agents Ivosidenib,Enasidenib, venetoclax, or a FLT3 inhibitor to fully understand thepharmacological response at each dose/schedule evaluated. (4) Toevaluate additional exploratory molecular, cellular and/or metabolicmarkers to enable prognostic and/or predictive associations withefficacy and/or resistance in each of the treatment arms

Study endpoints are displayed below in Table 3.

TABLE 3 Study endpoints for the study. Endpoint Name DescriptionTimeframe Primary MTD/MAD Review of dose-limiting toxicitiesApproximately (DLTs) and safety by DRT. 8 months Safety/tolerabilityType, frequency, seriousness and severity Approximately of AEs, andrelationship of AEs to study 13 months treatment. SecondaryPharmacokinetics To evaluate the pharmacokinetics (PK) Approximately oforal 5-azacytidine and each 13 months combination drug Overall ResponseRate of CR + CRi + MLFS + PR Approximately Rate (as assessed accordingto ELN 2017 AML response 13 months by the investigator) criteria. CR +CRi, CRh CR + CRi rate. Approximately CRi defined as response of bonemarrow 13 months blast <5% but with absolute neutrophil count (ANC) < 1× 10⁹/L or platelet < 100 × 10⁹/L CRh partial hematologic recovery:defined as less than 5% blasts in a BM aspirate sample with marrowspicules plus ANC > 0.5 × 10⁹/L and platelet count > 0.50 × 10⁹/L. Timeto response Time from first dose of study drug to first Approximatelydocumented CR/CRi/MLFS/PR 13 months according to criteria Duration ofTime from the first documented Approximately Response MLFS/CR/CRi/PR todocumented 13 months morphologic relapse, progression according to 2017ELN response criteria, or death due to any cause, whichever occurs firstEvent-Free Survival Time from randomization to documented Approximatelymorphologic relapse, progression 13 months according to 2017 ELNresponse criteria, or death from any cause, whichever occurs first.Overall Survival Time from randomization to death due to Approximatelyany cause. 30 months One-year survival The probability of survival at 1year from Approximately rate randomization 30 months CR without CR withnegativity for a genetic marker Approximately minimal residual byRT-qPCR, or CR with negativity by 8 months disease (CRMRD−) multi-colorflow cytometry Hematologic Rate of HI-N + HI-P + HI-E according toApproximately improvement rate IWG MDS HI criteria. 8 months ExploratoryPharmacodynamics To evaluate the pharmacodynamics (PD) Approximately ofeach combination agent to associate 8 months drug exposure with extentof target modulation and/or efficacy/tolerability. (in each treatmentcohort during dose/schedule escalation to RP2D) Correlative studiesEvaluation of molecular, cellular and Approximately metabolic markers inperipheral blood 8 months (PB) and/or bone marrow (BM) which may bepredictive of antitumor activity and/or resistance. Utilizing both pre-and post-treatment bone marrow biopsy samples acquired for purposes ofdisease assessment, change in baseline of candidate biomarkers will alsobe assessed using molecular/cell based assays ie, multi- parametric flowcytometry, DNA (gene sequencing), RNA expression (Nanostring or RNAseq),and/or in vitro assays. For example (but not limited to), MRD: flowcytometry or gene/PCR based assays (VAF); Gene mutationcharacterization: using next generation sequencing at baseline, duringtreatment, to relapse. Oral 5-azacytidine: DNA methylation changes inwhole blood/PBMC/PBLs or BM Ivosidenib and Enasidenib (IDH1/2): αKG and2-HG levels Venetoclax: BCL-2 expression/ amplification levels andcharacterization of other Bcl-2 family members, or Bcl-2 mimetic assaysMidostaurin and Gilteritinib: pFLT3/FLT3, pSTAT5/STAT5

Study Design This is an open-label, Phase I, multicenter umbrella trialto evaluate the safety and tolerability of oral 5-azacytidine as abackbone in combination with biomarker directed and novel therapies aslisted below:

-   -   Combination Arm A: oral 5-azacytidine+Ivosidenib in AML,        patients with IDH1 mutation,    -   Combination Arm B: oral 5-azacytidine+Enasidenib in AML patients        with IDH2 mutation,    -   Combination Arm C: oral 5-azacytidine+a FLT3 inhibitor in AML        patients with a FLT3 ITD or TKD mutation, and    -   Combination Arm D: oral 5-azacytidine+venetoclax in AML        patients.

The study population consists of AML patients who are in first relapse,refractory to 1 or 2 standard induction treatments, or newly diagnosedAML patients who are not candidates to receive intensive IC. The studycomprises of a Pre-screening Phase, Screening Phase, Treatment Phase anda Follow-up Phase.

Pre-Screening Phase All patients will sign a pre-screening consent forthe collection and determination of specific AML mutation results. Forpatients who have recent local hematopathology and gene mutation testingavailable from bone marrow aspirate and/or peripheral blood samplessince the time of relapse (or for newly diagnosed patients), thoseresults will be used for screening. For patients without localhematopathology and gene mutation testing, a referral lab testing forspecific AML mutations will be made available.

Patients will be assigned to the appropriate Combination Arm based onmutation testing results. All other AML patients without a specific AMLmutation (IDH1/2 or FLT3 wild-type) or if the specific mutation Arm isfilled/closed, and; who meet all eligibility criteria may enroll in oral5-azacytidine+venetoclax Combination Arm (Arm D) until this arm iscompleted/closed.

Note: In the rare case in which an eligible patient has multiplemutations, assignment to the potential treatment combination will bebased on joint investigator and medical monitor decision and documentedin the source document. For example: A patient with both IDH1 and FLT3mutations may be assigned to either the oral Ivosidenib or FLT3treatment arms.

Screening Phase Following pre-screening, and confirmation of specificAML mutation eligible for study entry, all enrolled patients willundergo screening procedures during the screening period i.e., within 28days prior to the start of study treatment. This will help determinestudy eligibility based on all eligibility criteria defined in theprotocol.

Treatment Phase Upon confirmation of eligibility, patients will beenrolled and begin treatment in the appropriate Combination Arm.Combination Arms and specific dosing levels will be made available forenrollment as per the sponsor.

Study treatment should be initiated on Day 1 of each treatment cycle.Treatment cycles are 28 days in duration. Visit windows are ±1 day inCycles 1 and 2; and starting in Cycle 3, windows are ±3 days.

During the treatment phase, the Dose Determination for each CombinationArm and dose level will be examined.

Dose Determination For the primary objective of determining the MTD/MADfor each combination arm during “dose determination”, a modifiedtoxicity probability interval (mTPI-2) method will be used. The mTPI-2method relies upon a statistical probability algorithm, calculated usingall patients treated in prior and current cohorts at the same dose levelto determine where future cohorts should involve dose re-escalation,stay in current dose, or dose de-escalation. Planned doses for eachCombination being tested are listed in Table 4. The dose determinationrules based on the mTPI-2 method are illustrated in Table 5.

A Dose Review Team (DRT) will review all adverse events (AEs)experienced by patients during Cycle 1 of each dose level to determinethe safety and tolerability of oral 5-azacytidine when administered incombination with Ivosidenib, Enasidenib, a FLT3 inhibitor, andvenetoclax.

Starting dose levels are based on currently used doses in larger trialsfor oral 5-azacytidine, Ivosidenib, and a FLT3 inhibitor; and approveddoses for combination agents of Enasidenib, a FLT3 inhibitor, andvenetoclax. Intermediate dose levels and adjustments to dosing may bediscussed and modified by the DRT. De-escalation to Dose Level −1A or−1B, will be based on the available safety information and attributionof the toxicity to either oral 5-azacytidine (to Level −1A) or thecombination agent (Level −1B). If toxicity is attributed to both drugsor unable to be assessed, both dose levels may be opened upon discussionwith the DRT. Intrapatient dose adjustments for oral 5-azacytidine mayoccur after discussion with the Celgene medical monitor up to 300 mg QDfor 21 days.

The first group of patients enrolled at a new dose level will begintreatment in a staggered manner as determined by the sponsor.

TABLE 4 Dose Determination Starting Dose Levels for Combination ArmsCombination Combination Combination Combination A: oral 5- B: oral 5- C:oral 5- D: oral 5- azacytidine + azacytidine + azacytidine +azacytidine + Ivosidenib Enasidenib Midostaurin venetoclax Dose Level +1oral 5- oral 5- oral 5- oral 5- azacytidine azacytidine azacytidineazacytidine 300 mg QD x 300 mg QD x 300 mg QD x 300 mg QD x 21 d + 21d + 21 d + 21 d + Ivosidenib Enasidenib Midostaurin Venetoclax 500 mg QD100 mg QD 50 mg BID 400 mg QD Starting Dose oral 5- oral 5- oral 5- oral5- (Dose Level 0) azacytidine azacytidine azacytidine azacytidine 300 mgQD x 300 mg QD x 300 mg QD x 300 mg QD x 14 d + 14 d + 14 d + 14 d +Ivosidenib Enasidenib Midostaurin Venetoclax 500 mg QD 100 mg QD 50 mgBID 400 mg QD Dose Level −1A oral 5- oral 5- oral 5- oral 5- azacytidineazacytidine azacytidine azacytidine 200 mg QD x 200 mg QD x 200 mg QD x200 mg QD x 14 d + 14 d + 14 d + 14 d + Ivosidenib EnasidenibMidostaurin Venetoclax 500 mg QD 100 mg QD 50 mg BID 400 mg QD DoseLevel −1B oral 5- oral 5- oral 5- oral 5- azacytidine azacytidineazacytidine azacytidine 300 mg QD x 300 mg QD x 300 mg QD x 300 mg QD x14 d + 14 d + 14 d + 14 d + Ivosidenib Enasidenib Midostaurin Venetoclax250 mg QD 50 mg QD 25 mg BID 300 mg QD Dose Level −2 oral 5- oral 5-oral 5- oral 5- azacytidine azacytidine azacytidine azacytidine 200 mgQD x 200 mg QD x 200 mg QD x 200 mg QD x 14 d + 14 d + 14 d + 14 d +Ivosidenib Enasidenib Midostaurin Venetoclax 250 mg QD 50 mg QD 25 mgBID 300 mg QD

A cycle is defined as 28 days. Study visits will occur weekly for thefirst two cycles, then every two weeks (Day 1 and Day 15) starting fromCycle 3. After dose determination is complete for each combination arm,all ongoing patients will continue to receive the same dose andtreatment until discontinuation.

Modified Toxicity Probability Interval (mTPI-2). During dosedetermination, a modified toxicity probability interval (mTPI-2) methoddesign will be used to determine the Maximum Tolerated Dose (MTD) orMaximum Administered Dose (MAD) for the combination. The MTD is definedas the highest dose level evaluated that does not exceed the target DLTprobability of 0.30 in the first cycle of the treatment. The mTPI-2determination is independent for every dose level in each CombinationArm. The dose determination rules based on the mTPI-2 method areillustrated in Table 5.

TABLE 5 mTPI-2 Dose Determination Rules 3 4 5 6 7 8 9 10 11 12 0 E E E EE E E E E E 1 S S E E E E E E E E 2 D D D S S S E E E E 3 DU DU D D D DS S S S 4 DU DU DU D D D D D S 5 DU DU DU DU DU D D D 6 DU DU DU DU DUDU D 7 DU DU DU DU DU DU 8 DU DU DU DU DU 9 DU DU DU DU 10 DU DU DU 11DU DU 12 DU Target toxicity probability = 0.3, ε₁ = 0.05, ε₂ = 0.05 *Column indicates the number of patients treated. Row indicates thenumber of patients with DLTs * E: Escalate to the next higher dose; S:Stay at the same dose; D: De-escalate to the previous lower dose; DU:De-escalate to the previous lower dose and the current dose will neverbe used again in the trial.

In general, the rules for dose-finding using the mTPI-2 method includethe following:

-   -   Each Cohort within a Dose Level Arm will enroll 2-6 patients;        (Note: at least 3 patients should be enrolled and treated in the        first cohort at a new dose level)    -   When at least 3 patients are evaluated for DLT at the current        dose level or experience a DLT, the next cohort will be enrolled        and assigned to the dose level per Table 4. All patients        evaluated at the same dose level, including prior cohort(s) and        current cohort, will be used to determine the dose level of the        next cohort.    -   If a patient does not receive the protocol-specified        administrations of the study medication within the DLT        observation period for reasons other than study drug-related        toxicity, another patient will be enrolled as a replacement at        the current dose level.    -   The determination rules are independent for each Dose Level in        every Combination Arm.    -   The dose-finding phase for each combination will conclude when        at least 10 patients have been treated at a dose level that is        predicted to be the MTD or doses appear to be overly toxic and        the MTD cannot be determined in the current trial.    -   Patients must complete at least 80% of the planned total dose of        oral 5-azacytidine and the combination agent, or experience a        DLT during DLT observation period, in order to be considered        evaluable for DLT. The dose escalation decision rules are        defined in the Statistical Methods Section.

The DLT observation period for evaluation for each dose level is thefirst cycle (28 days) of treatment of the current dose level, or up tothe DLT if it occurs earlier.

The following is an example of dose finding procedure for CombinationArm B (oral 5-azacytidine+Enasidenib). The four predetermined doselevels are:

-   -   Dose Level+1: oral 5-azacytidine 300 mg QD Days 1-21+Enasidenib        100 mg QD Days 1-28;    -   Starting Dose Level 0: oral 5-azacytidine 300 mg QD Days        1-14+Enasidenib 100 mg QD Days 1-28;    -   Dose Level −1A: oral 5-azacytidine 200 mg QD Days        1-14+Enasidenib 100 mg QD Days 1-28;    -   Dose Level −1B: oral 5-azacytidine 300 mg QD Days        1-14+Enasidenib 50 mg QD Days 1-28;    -   Dose Level −2: oral 5-azacytidine 200 mg QD Days 1-14+Enasidenib        50 mg QD Days 1-28.

The dose-finding will start with enrollment of 3 patients and treatmentat the Starting Dose Level (Dose Level 0): oral 5-azacytidine 300 mg QDDays 1-14+Enasidenib 100 mg QD Days 1-28. By the end of the DLTobservation period of the 3 patients, the next cohort will be enrolledand assigned to the dose level which is determined based on the numberof DLTs and the mTPI-2 rule table (Table 5):

-   -   If 3/3 DLTs—De-escalate to Dose Level −1A or −1B (depending on        the attributed agent), enroll and assign this new dose level        cohort with 3 patients. The current or higher dose is too toxic        and cannot be explored again.    -   If 2/3 DLTs—De-escalate to Dose Level −1A or −1B, enroll and        assign this new dose level cohort with 3 patients.    -   If 1/3 DLT—Stay at the same dose level (Dose Level 0), enroll        additional 2 patients (2^(nd) cohort). Note the total number of        patients at Dose Level 0 becomes 5.    -   If 0/3 DLT—Stay at the same dose level (Dose Level 0) with an        option to escalate (if applicable), enroll an additional 2        patients (2^(nd) cohort). If the dose is escalated, assign a new        dose level cohort with 3 patients.

If a new Dose Level is started, the mTPI-2 rule table resets and the newdose cohort level is evaluated separately from the prior Dose Level.

At the end of the DLT observation period of the 2^(nd) cohort for a doselevel, the next cohort (3^(rd) cohort) will be enrolled and assigned tothe dose level which is determined based on the number of DLTs and themTPI-2 rule table (Table 4). All patients and their DLTs at the samedose level will be used, including the current cohort and any priorcohort(s).

Except for requiring at least 3 patients in the first cohort at eachdose level, the size of the additional cohorts in a dose level can beflexible. For example, if the dose level continues at the same level perthe mTPI-2 rules, the sequence of the cohort sizes could be 3, 2, 2, 3,etc.

The dose-finding will conclude when at least 10 patients have beentreated at a dose that is predicted to be the MTD or if doses appear tobe overly toxic and the MTD cannot be determined in the currentcombination arm for the study.

After determination of the MTD/MAD, the DRT will review all safety datafor patients who were treated in the combination arm (Arms A through E)to determine the RP2D for the Combination Arm which may be used in anexpansion cohort.

End of Treatment (EOT): Treatment will continue until documented diseaseprogression, unacceptable adverse event(s), intercurrent illness thatprevents further administration of treatment, investigator's decision towithdraw the patient, patient withdraws consent, pregnancy of thepatient, noncompliance with trial treatment or procedure requirements,or administrative reasons.

At the EOT visit, an evaluation will be performed with a visit window of±7 days. If the EOT occurs during a scheduled visit, all EOT assessmentsmust also be completed.

Follow-up Phase After the end of treatment, each patient will befollowed for 28 days for AE monitoring. Patients who discontinuetreatment for reasons other than disease progression will havepost-treatment follow-up for disease status until disease progression,initiating a non-study cancer treatment, withdrawing consent, orbecoming lost to follow-up.

The Follow-up Phase will have assessments every 28 days for the firstyear then every 3 months thereafter.

In addition, all patients will be followed by telephone contact forsurvival until death, withdrawal of consent or the end of the trial,whichever comes first.

Study Population/Estimated No. Patients Up to approximately 72 patientswill be enrolled in this study (4 Combination Arms×18 patients each).

For each Combination Arm dose level, approximately 12 patients may beenrolled to evaluate safety, tolerability and preliminary efficacy.Depending on the dose level used for dose determination, up to 18patients may be enrolled for each Combination Arm.

Additional patients for dose expansion may also be enrolled aftercompletion of the primary objective for each Combination Arm.

Key Inclusion Criteria Patients must satisfy the following criteria tobe enrolled in the study:

-   -   1. Patient is ≥18 years of age at the time of signing the        informed consent form (ICF).    -   2. Patients with AML as defined by the WHO Classification:        -   a. First relapse (i.e. recurring after having achieved an            initial response [CR/CRi] to chemotherapy except            promyelocytic leukemia [APML]); or        -   b. Persisting/refractory after 1 to 2 primary induction            courses (ie, no response after 1 to 2 prior chemotherapy            regimens); or        -   c. Newly diagnosed AML who are not candidates for intensive            chemotherapy    -   3. Patients must have the following AML specific mutation in        bone marrow aspirate and/or peripheral blood sample documented        since last progression/relapse (or recent testing for newly        diagnosed):        -   a. For Combination Arm −A (IDH1): Have confirmation of an            IDH1 mutation        -   b. For Combination Arm −B (IDH2): Have confirmation of an            IDH2 mutation        -   c. For Combination Arm −C(FLT3): Have confirmation of a FLT3            mutation (ITD or TKD)        -   d. For Combination Arm D (BCL-2): Patients without any of            the specific mutations listed or have a mutation in a closed            combination arm above may be enrolled into this oral            5-azacytidine+venetoclax arm.    -   4. Patient has Eastern Cooperative Oncology Group (ECOG)        performance status of 0, 1 or 2    -   5. Patient has adequate organ function defined as:        -   Aspartate aminotransferase (AST)/serum glutamic oxaloacetic            transaminase (SGOT) and alanine aminotransferase (ALT)/serum            glutamic pyruvic transaminase (SGPT)≤2.5× upper limit of            normal (ULN), unless considered due to leukemic organ            involvement        -   Serum total bilirubin≤1.5×ULN, unless considered due to            Gilbert's syndrome (eg, a gene mutation in UGT1A1) or            leukemic organ involvement        -   Creatinine clearance>30 mL/min based on the Modification of            Diet in Renal Disease (MDRD) glomerular filtration rate            (GFR):

GFR(mL/min/1.73 m2)=175×(serum creatinine)−1.154×(Age)−0.203×(0.742 iffemale)×(1.212 if African American)

-   -   6. Agree to serial bone marrow aspirate/biopsies.    -   7. Females of childbearing potential (FCBP)* must:        -   Have two negative pregnancy tests as verified by the            Investigator prior to starting study therapy. She must agree            to ongoing pregnancy testing during the course of the study,            and after end of study therapy. This applies even if the            patient practices true abstinence* from heterosexual            contact.        -   Either commit to true abstinence* from heterosexual contact            (which must be reviewed on a monthly basis and source            documented) or agree to use, and be able to comply with            highly effective contraception without interruption, −3 days            prior to starting investigational product, during the study            therapy (including dose interruptions), and for 90 days            after discontinuation of study therapy, or longer if            required for each compound and/or by local regulations.            -   For Combination Arm A: the timeframe is extended to 4                (four) months following the last study treatment dose            -   For Combination Arm B: the timeframe is extended to 4                (four) months following the last study treatment dose            -   For Combination Arm D: the timeframe is extended to 6                (six) months following the last study treatment dose.    -   8. Male patients must practice true abstinence* (which must be        reviewed on a monthly basis) or agree to use a condom during        sexual contact with a pregnant female or a female of        childbearing potential while participating in the study, during        dose interruptions and for at least 90 days following        investigational product discontinuation, or longer if required        for each compound and/or by local regulations, even if he has        undergone a successful vasectomy.        -   For Combination Arm A: the timeframe is extended to 4 (four)            months following the last study treatment dose        -   For Combination Arm B: the timeframe is extended to 4 (four)            months following the last study treatment dose        -   For Combination Arm D: the timeframe is extended to 6 (six)            months following the last study treatment dose.    -   9. Patient must understand and voluntarily sign an ICF prior to        any study-related assessments/procedures being conducted.    -   10. Patient is willing and able to adhere to the study visit        schedule and other protocol requirements.

Exclusion Criteria The presence of any of the following will exclude apatient from enrollment:

-   -   1. Patient is suspected or proven to have acute promyelocytic        leukemia based on morphology, immunophenotype, molecular assay,        or karyotype    -   2. Patient has AML, secondary to chronic myelogenous leukemia or        patient is BCR-ABL1 t(9; 22)(q34;q11) positive.    -   3. Patient has a white blood cell count of >15,000 cells/mcl        (15×10⁹/L). Note: Hydroxyurea is allowed up to 72 hours prior to        the screening blood draw and limited use only during Cycle 1 for        cytoreduction (to control high white blood cell count).    -   4. Patient has received systemic anticancer therapy or        radiotherapy<28 days prior to the start of study treatment. Note        that hydroxyurea is allowed prior to the start of study        treatment for the control of leukocytosis.    -   5. Patient has received therapy with hypomethylating agents for        AML and did not achieve CR/CRi/PR/MLFS/SD during first line        therapy    -   6. Patient has received investigational agents<30 days or 5        half-lives, whichever is longer, prior to the start of study        treatment    -   7. Patient has undergone HSCT within 90 days prior to the start        of study treatment, or on immunosuppressive therapy post HSCT at        the time of screening, or with graft-versus-host disease (GVHD).        The use of a stable dose of oral steroid post-HSCT, or topical        steroids for ongoing skin GVHD is permitted.    -   8. Patient has persistent, clinically significant        non-hematologic toxicities from prior therapies which have not        recovered to <Grade 2    -   9. Patient has or is suspected of having central nervous system        (CNS) leukemia. Evaluation of cerebrospinal fluid is only        required if CNS involvement by leukemia is suspected during        screening.    -   10. Patient has active uncontrolled systemic fungal, bacterial,        or viral infection (defined as ongoing signs/symptoms related to        the infection without improvement despite appropriate        antibiotics, antiviral therapy, and/or other treatment). The        patient should be afebrile for at least 72 hours.    -   11. Patient has immediate life-threatening, severe complications        of leukemia such as uncontrolled bleeding, pneumonia with        hypoxia or shock, and/or disseminated intravascular coagulation    -   12. Patient has prior history of malignancy, other than MDS, MPN        or AML, unless the patient has been free of the disease for ≥1        year prior to the start of study treatment.        -   However, patients with the following history/concurrent            conditions are allowed:            -   Basal or squamous cell carcinoma of the skin            -   Carcinoma in situ of the cervix            -   Carcinoma in situ of the breast            -   Incidental histologic finding of prostate cancer (T1a or                T1b using the tumor, nodes, metastasis clinical staging                system)    -   13. Patient is known seropositive or active infection with human        immunodeficiency virus (HIV), or active infection with hepatitis        B virus (HBV) or hepatitis C virus (HCV)    -   14. Patient is known to have dysphagia, short-gut syndrome,        gastroparesis, or other conditions that limit the ingestion or        gastrointestinal absorption of drugs administered orally    -   15. Patients has uncontrolled hypertension (systolic blood        pressure [BP]>180 mmHg or diastolic BP>100 mmHg) or has not been        stable for at least 1 month prior to treatment    -   16. Significant active cardiac disease within the previous 6        months prior to signing the ICF, including:        -   New York Heart Association (NYHA) Class III or IV congestive            heart failure;        -   Unstable angina or angina requiring surgical or medical            intervention;        -   Significant cardiac arrhythmia; and/or        -   Myocardial infarction    -   17. Patient is a pregnant or lactating female    -   18. Patient has known or suspected to have hypersensitivity to        any of the components of the assigned study treatments    -   19. Patient has any significant medical condition, laboratory        abnormality, or psychiatric illness that would prevent the        patient from participating in the study    -   20. Patient has any condition including the presence of        laboratory abnormalities, which places the patient at        unacceptable risk if he/she were to participate in the study    -   21. Patient has any condition that confounds the ability to        interpret data from the study    -   22. Additional exclusion criteria for patients based on the        planned Combination Arm are:        -   a. For Combination Arm −A (IDH1):            -   Patient has QTc interval (ie, Fridericia's correction                [QTcF])≥450 ms or other factors that increase the risk                of QT prolongation or arrhythmic events (eg, heart                failure, hypokalemia, family history of long QT interval                syndrome) at screening            -   Patient is taking those medications that are known to                prolong QT interval unless the patient can be                transferred to other medications at least 5 half-lives                prior to the start of study treatment            -   Patient has significant active cardiac disease within 6                months prior to the start of study treatment; acute                coronary syndrome (AC S); and/or stroke; or left                ventricular ejection fraction (LVEF)<40% by                echocardiogram (ECHO) or multi-gated acquisition (MUGA)                scan obtained within 28 days prior to the start of study                treatment        -   b. For Combination Arm −B (IDH2):            -   Patient is taking the breast cancer resistance protein                (BCRP) transporter-sensitive substrate rosuvastatin                should be excluded from the study unless the patient can                be transferred to other medications at least 5                half-lives prior to the start of study treatment            -   Patient is taking the following sensitive CYP substrate                medications that have a narrow therapeutic range are                excluded from the study unless the patient can be                transferred to other medications at least 5 half-lives                prior to the start of study treatment: paclitaxel and                docetaxel (CYP2C8), phenytoin (CYP2C9), S-mephenytoin                (CYP2C19), thioridazine (CYP2D6), theophylline, and                tizanidine (CYP1A2)        -   c. For Combination Arm −C(non-specific FLT3):            -   Patients with hypokalemia and hypomagnesemia at                Screening (values below lower limit of normal [LLN]).            -   Patient has a history of congestive heart failure NYHA                class 3 or 4, unless a screening echocardiogram                performed within 3 months prior to study entry results                in a left ventricular ejection fraction that is ≥45%.            -   Patients with mean of triplicate Fridericia-corrected QT                interval (QTcF)>450 ms at Screening.            -   Patients with Long QT Syndrome at Screening.            -   Patient requires treatment with concomitant drugs that                are strong inducers of cytochrome P450 (CYP)3A.            -   Patient requires treatment with concomitant drugs that                are strong inhibitors or inducers of P glycoprotein                (P-gp) with the exception of drugs that are considered                absolutely essential for the care of the patient.            -   Patient requires treatment with concomitant drugs that                target serotonin 5-hydroxytryptamine receptor 1 (5HT1R)                or 5-hydroxytryptamine receptor 2B (5HT2BR) or sigma                nonspecific receptor with the exception of drugs that                are considered absolutely essential for the care of the                patient.        -   d. For Combination Arm −D (BCL-2):            -   Received strong CYP3A inhibitors, moderate CYP3A                inhibitors, strong CYP3A inducers, moderate CYP3A                inducers, strong CYP2C8 inhibitors, CYP2C8 substrates,                or OATP1B1/3 substrates within 7 days prior to                initiation of study treatment            -   Received strong CYP2C8 inducers within 14 days prior to                initiation of study treatment

Length of Study The full length of the study is expected to beapproximately 30 months including recruitment, screening, treatment, andfollow up. For a single patient, the expected duration of the study isapproximately 8 months, including a screening period phase of up to 28days.

The End of Trial is defined as either the date of the last visit of thelast patient to complete the post-treatment follow-up, or the date ofreceipt of the last data point from the last patient that is requiredfor primary, secondary, and/or exploratory analysis, as pre-specified inthe protocol, whichever is the later date.

Overview of Key Efficacy Assessments Response to treatment will beassessed by the investigators according to the European Leukemia Net(ELN) AML Response Criteria (Dohner, H et al., Blood, 2017 Jan. 26,129(4): 424-47). Hematologic improvement (HI) in patients with newlydiagnosed AML will also be assessed according to the IWG myelodysplasticsyndromes HI criteria (Cheson, 2006). Patients are to undergoend-of-treatment evaluations when study treatment is discontinued. Thereason for treatment discontinuation will be recorded in the electroniccase report form (eCRF) pages and in the source document.

At baseline, a bone marrow aspirate and biopsy sample are required.Serial blood and bone marrow aspirate sampling (with bone marrow biopsyif needed) will be used to determine response to therapy starting atCycle 2 Day 1 and will be assessed every other cycle (Cycle 4, Cycle 6,etc) until remission (or EOT if no remission). At remission, anadditional bone marrow aspirate and biopsy are required. Duringremission, an aspirate should be collected every 4 months or whenclinically indicated. At EOT, an additional bone marrow aspirate will becollected. Response will be assessed locally according to 2017 ELNcriteria based on reported hematology laboratory parameters, peripheralblood smear, bone marrow aspirates and/or biopsies. The site needs toensure peripheral blood and BMA/BMB samples are collected and stored forexploratory testing at the time of every bone marrow collection.

Patients who discontinue study treatment prior to relapse or progressionwill complete monthly site visits until confirmation of relapse orprogression. For patients who have discontinued study treatment due torelapse or progression, monthly follow up can be performed by sitevisits or phone calls. Patients will be followed until they have died,are lost to follow up, withdraw consent for further data collection, oruntil study closure.

Overview of Key Safety Assessments All patients will be monitored forAEs during the study. Adverse events will be graded in severityaccording to the guidelines outlined in the NCI Common TerminologyCriteria for Adverse Events (CTCAE) version 5.

Dose Limiting Toxicities (DLTs) Toxicity severity will be gradedaccording to the Common Terminology Criteria for Adverse Events (CTCAE)version 4.03. DLTs during dose determination will be based on adverseevents that occur during the DLT observation period. For this study, theDLT observation period is defined as the first treatment cycle (28 days)and patients will be evaluated at the completion of each cohort at adose level. If DLT events occurs during the 2nd treatment cycle, no newpatients may begin treatment until review and discussion by the DRT.Events that are considered DLTs based on their duration will beconsidered a DLT if they start during the DLT assessment period.

Any of the following events will be considered a DLT unless the eventcan be attributed by the investigator to a clearly identifiable causesuch as underlying illness or disease progression, other concurrentillness, or concomitant medication:

-   -   A hematological DLT is defined as non-recovery from a Grade 4        hematological toxicity within 42 days from Day 1 of the previous        cycle.        -   For the purpose of DLT determination, the Grade 4            hematological toxicity is defined as: (i) ANC<0.5×10⁹/L            (Grade≥4) and/or (ii) PLT count<25.0×10⁹/L (Grade≥4)        -   A hematological DLT can only be assigned if the delay in            recovery per investigator's judgement is due to            treatment-induced toxicity rather than residual effect of            AML (a bone marrow sample may be indicated to document that            this is not due to AML with bone marrow blasts<5%).            -   For the purpose of DLT determination, recovery from                hematological toxicity is defined as an increase in cell                counts from nadir by ≥50% of the reduction from baseline                observed at the time of nadir            -   The use of G-CSF or platelet transfusions are prohibited                during the DLT evaluation period. Patients receiving                these treatments will be determined as not evaluable and                will be replaced.            -   Patients who enter study with low hematology counts                (Grade 3 and Grade 4), will not be considered evaluable                for a hematologic DLT unless a clinically meaningful                change has occurred (non-recovery of counts within 42                days of Day 1 of the previous cycle and a mandatory bone                marrow assessment indicates a treatment induced bone                marrow toxicity).    -   A non-hematological DLT is defined as the occurrence of a        Grade≥3 clinically significant non-hematologic adverse event or        abnormal laboratory value occurring during the first cycle on        study that cannot be attributed by the investigator to a clearly        identifiable cause such as disease progression, underlying        illness, concurrent illness, or concomitant medication, with the        following EXCEPTIONS:        -   Grade 3 fatigue        -   Grade 3 nausea, vomiting, or diarrhea that can be managed to            ≤Grade 2 (or baseline) with standard antiemetic or            antidiarrheal medications within 72 hours        -   For IDH1 or IDH2 combination: Grade 3 or 4 leukocytosis, or            Grade 3 differentiation syndrome (DS), that can be managed            to <Grade 3 with hydroxyurea and/or corticosteroids        -   Grade 3 Tumor Lysis Syndrome unless controlled by medical            management

For any DLT occurring during the DLT observation period which mayrequire interruption of both oral 5-azacytidine and/or the combinationagent, reintroduction at a reduced dose after the toxicity grade hasreturned to Grade≤1 or to baseline may occur based on discussion withthe Celgene Medical Monitor. The start of the subsequent cycle may needto be delayed to allow for recovery from a DLT or any other adverseevent. A dose modification schedule will be available for eachcombination arm as guidance. Patients with neutropenia orthrombocytopenia as a consequence of the disease prior to the start oftherapy do not require treatment interruptions for myelosuppression.However, the investigator may use clinical judgement based on patient'sdisease status, clinical condition, and comorbidities to make thedecision regarding treatment modifications. Dose reductions of the studytreatment in these patients should be considered on an individual casebasis and discussed with the Medical Monitor.

Any reduced Dose Level of oral 5-azacytidine and/or the combinationagent will be jointly defined by the investigator and the CelgeneMedical Monitor (MM). The dose may be increased thereafter upon jointdetermination of the investigator and the Celgene MM. All decisionsregarding continued dosing for individual patients with DLT will bemedically managed by the investigator, and per discussion with theCelgene MM, as appropriate.

All patients are allowed to receive antibiotic prophylaxis (prohibitedmedication excluded) per standard institution guidelines during the DLTobservation period.

Criteria for Evaluation Safety evaluations include, but are not limitedto capturing AEs, clinical laboratory testing (hematology andchemistry), physical examinations, vital sign measurements,electrocardiogram (ECG) testing, as a measure of safety and tolerabilityfor the entire study duration.

For each Combination Arm, dose escalation/de-escalation decisions duringdose determination will be guided by assessment of DLTs and based on thecumulative number of patients who experience a DLT at a given doselevel.

Study Treatments Oral 5-azacytidine will be administered orally oncedaily on Days 1-14 (or 21) of each 28-day treatment cycle. Patientsshould be instructed to take their daily dose at approximately the sametime each day. Each dose should be taken with approximately 240 mL (8ounces) of room temperature water. Oral 5-azacytidine may be taken on anempty stomach or with food.

Ivosidenib is administered orally once a day (QD) on Days 1-28 of each28-day cycle. Patients should be instructed to take their daily dose atapproximately the same time each day±6 hours. Each dose should be takenwith a glass of water and consumed over as short a time as possible.Patients should be instructed to swallow tablets whole and to not chewthe tablets.

Enasidenib is administered orally once a day (QD) on Days 1-28 of each28-day cycle. Patients should be instructed to take their daily dose atapproximately the same time each day±6 hours. Each dose should be takenwith a glass of water and consumed over as short a time as possible.Patients should be instructed to swallow tablets whole and to not chewthe tablets. Fasting is required for 2 hours prior to and 1 hourfollowing Enasidenib administration. Water is allowed during fasting.

Midostaurin is administered orally twice daily (BID) on Days 1-28 ofeach 28-day cycle. Patients should be instructed to take their doses atapproximately the same time each day±Y hours. Each dose should be takenwith a meal and water, and consumed over as short a time as possible.Patients should be instructed to swallow tablets whole and to not chewthe tablets

Gilteritinib is administered orally once a day (QD) on Days 1-28 of each28-day cycle. Patients should be instructed to take their daily dose atapproximately the same time each day±Y hours. Each dose should be takenwith a meal and water, and consumed over as short a time as possible.Patients should be instructed to swallow tablets whole and to not chewthe tablets.

Venetoclax is administered orally once a day (QD) on Days 1-28 of each28-day cycle. A brief dose ramp-up occurs for Cycle 1 with the dosing of100 mg on Day 1, 200 mg on Day 2, and 400 mg on Day 3. Patients shouldbe instructed to take their daily dose at approximately the same timeeach day±6 hours. Each dose should be taken with a meal and water, andconsumed over as short a time as possible. Patients should be instructedto swallow tablets whole and to not chew the tablets.

In cases of dose interruption/modification, cycle visits will continueon the same schedule and timing (e.g. Cycle 3 is 28 days after Cycle 2,etc). If a drug interruption occurs, the other study drug may continuethe treatment schedule.

Statistical Methods At the end of the dose determination, theisotopically transformed DLT rate which is closest to the target DLTrate will be selected as the estimated MTD/MAD. If two or more doses areequally close to the target DLT rate, the highest dose among the tieddoses will be chosen if they are less than the target DLT rate, or thelowest dose among the tied doses will be chosen if they are higher thanthe target DLT rate.

Statistical analyses will be primarily descriptive in nature.Tabulations will be produced for disposition, demographic and baselinedisease characteristics, safety, PK, PD, and clinical activityparameters. Categorical data will be summarized by frequencydistributions (numbers and percentages of patients) and continuous datawill be summarized by descriptive statistics (mean, standard deviation,median, minimum, and maximum). Data will be summarized by dose level andoverall where appropriate.

The results of the clinical study will show that: (1) oral 5-azacytidinein combination with ivosidenib at the maximum tolerated dose (MTD)and/or maximum administered dose (MAD) is safe and tolerated in AMLpatients with an IDH1 mutation; (2) oral 5-azacytidine in combinationwith enasidenib at the maximum tolerated dose (MTD) and/or maximumadministered dose (MAD) is safe and tolerated in AML patients with anIDH2 mutation; (3) oral 5-azacytidine in combination with an FLT3inhibitor, such as midostaurin and gilteritinib, at the maximumtolerated dose (MTD) and/or maximum administered dose (MAD) is safe andtolerated in AML patients with a FLT3 ITD or TKD mutation; and/or (4)oral 5-azacytidine in combination with venetoclax at the maximumtolerated dose (MTD) and/or maximum administered dose (MAD) is safe andtolerated in AML patients.

Example 3

Study Objectives: The primary objective of the study is as follows.

Dose-finding (Phase 1b): To establish maximum tolerated dose (MTD) ormaximum administered dose (MAD) and/or the recommended Phase 2 dose(RP2D) by evaluating safety and tolerability of oral 5-azacytidine incombination with venetoclax or gilteritinib in patients with AML orhigher-risk MDS.

Dose expansion (Phase 2): To assess efficacy of oral 5-azacytidine whengiven in combination with venetoclax at the RP2D in newly diagnosedpatients with AML or higher-risk MDS.

The secondary objectives are as follows:

Dose-finding (Phase 1b): To assess the preliminary efficacy of oral5-azacytidine in combination with venetoclax or gilteritinib in patientswith AML or higher-risk MDS.

Dose expansion (Phase 2): To further evaluate safety and tolerability oforal 5-azacytidine when given in combination with venetoclax at the RP2Din newly diagnosed patients with AML or higher-risk MDS.

All parts (Phase 1b/2): To characterize the pharmacokinetics (PK) oforal 5-azacytidine combination with venetoclax or gilteritinib.

The exploratory objectives are as follows: All parts (Phase 1b/2): (1)To characterize the pharmacodynamics (Pd) to understand the mechanisticeffects of oral 5-azacytidine in combination with venetoclax orgilteritinib. (2) To evaluate the MRD response rate and MRD conversionrate by multicolor flow cytometry (MFC) and/or Next GenerationSequencing (NGS). (3) To explore the duration of MRD response byassessments of bone marrow aspirate (BMA) and examination of peripheralblood smears (PBS). (4) To investigate the relationship between PK, PDbiomarkers, and/or clinical outcomes of oral 5-azacytidine incombination with venetoclax or gilteritinib. (5) To evaluate molecularand/or cellular markers in the BM and blood that may be predictive ofefficacy of oral 5-azacytidine in each combination

Study endpoints are displayed below in Table 6.

TABLE 6 Study endpoints for the study. Endpoint Name DescriptionTimeframe AML Cohort - Phase 1b Primary MTD/MAD, Dose-limitingtoxicities (DLTs) From first dose (C1D1) to the and/or RP2D evaluatedusing NCI CTCAE end of Cycle 1 Version 5.0 Safety/ Type, frequency,seriousness and From informed consent form tolerability severity of AEs(using NCI (ICF) signature to 28 days CTCAE Version 5.0), and after lastdose of IP relationship of AEs to study treatment, clinical laboratoryevaluations Secondary Preliminary CR/CRh rate: defined as the ratePreliminary efficacy efficacy of achieving CR or CRh AML Cohort - Phase2 Primary Efficacy at CR/CRh rate: defined as the rate From first doseto 28 days RP2D of achieving CR or CRh after last dose Secondary Safety/Type, frequency, seriousness and From ICF signature to 28 daysTolerability severity of AEs (using NCI after last dose of IP CTCAEVersion 5.0), and relationship of AEs to study treatment, clinicallaboratory evaluations AML Cohort - Phase 1b and 2 Secondary EfficacyORR defined as the rate of From first dose to 28 days achieving CR, CRi,CRp, PR, or after last dose MLFS Response assessed by Investigatoraccording to the European Leukemia Net (ELN) AML Response Criteria(Döhner, H et al., Blood, 2017 Jan. 26, 129(4): 424-47) and IWG for AMLResponse Criteria (Cheson B. D. et al., J Clin Oncol. 2003, 21(24):4642- 92003). Time to response (CR, CRi, From first dose to first CRp,PR, MLFS) response CR, CRi, CRp, PR, MLFS DOR (CR/CRh) From firstresponse CR or CRh to first relapse or death, whichever occurs first DOR(CR/CRi/CRp/PR/ From first response (CR, CRi, MLFS) CRp, PR, or MLFS) tofirst relapse, disease progression, or death, whichever occurs firstEvent-Free Survival (EFS) From first dose to first relapse, diseaseprogression, or death from any cause, whichever occurs firstRelapse-Free Survival (RFS) From first response to relapse, death, orlast follow-up Exploratory MRD Rate of having at least a one log MRDassessments will be Response reduction in disease burden or an performedat Screening, Day 1 Rate MRD negative (10⁻³) test result of Cycles 2, 3,4, then every 2 cycles thereafter (ie, Cycles 6, 8, 10, etc) untilremission or EOT MRD Rate of achieving MRD MRD assessments will beConversion negativity (10⁻³) at any time on performed at Screening, Day1 Rate therapy of Cycles 2, 3, 4, then every 2 cycles thereafter (ie,Cycles 6, 8, 10, etc) until remission or EOT Duration of Evaluate thedurability of MRD Time from achieving first MRD response by serial bonemarrow MRD response to last MRD Response aspirate assessment for MRDnegative assessment or having a positive MRD result or increase indisease burden by at least one log. Death will be censored. MDS Cohort -Phase 1b Primary MTD/MAD, Dose-limiting toxicities (DLTs) From firstdose to the end of and/or RP2D evaluated using NCI CTCAE Cycle 1 Version5.0 Safety/ Type, frequency, seriousness and From ICF signature to 28days tolerability severity of AEs (using NCI after last dose of IP CTCAEVersion 5.0), and relationship of AEs to study treatment, clinicallaboratory evaluations Secondary Preliminary CR, PR, marrow (mCR):defined From first dose to 28 days efficacy as the rate of achieving CR,PR, after last dose mCR Response assessed by Investigator according toIWG for MDS Response Criteria (Cheson B. D. et al., Blood 2006; 108(2):419-425) MDS Cohort - Phase 2 Primary Efficacy at CR, PR, mCR: definedas the From first dose to 28 days RP2D rate of achieving CR, PR, mCRafter last dose Secondary Safety/ Type, frequency, seriousness and FromICF signature to 28 days Tolerability severity of AEs (using NCI afterlast dose of IP CTCAE Version 5.0), and relationship of AEs to studytreatment, clinical laboratory evaluations MDS Cohort - Phase 1b and 2Secondary Efficacy Time to response (CR, mCR, From first dose to firstPR) according to IWG Response response CR, mCR, PR Criteria for MDS(Cheson B. D. et al., Blood 2006; 108(2): 419- 425) DOR (CR, mCR, PR)From first response (CR, mCR, PR) to first relapse, disease progression,or death, whichever occurs first Event-Free Survival (EFS) From firstdose to first relapse, disease progression, or death from any cause,whichever occurs first Relapse-Free Survival (RFS) From first responseto relapse, death, or last follow-up AML and MDS Cohorts - Phase 1b and2 Secondary Efficacy Overall Survival (OS) From first dose to deathHematologic Rate of HI-N + HI-P + HI-E improvement according to IWG MDSHI rate (HI) criteria (Cheson B. D. et al., Blood 2006; 108(2): 419-425)One-year The probability of survival at 1 From first dose to survivalrate year from first dose approximately 12 months PK PK parameters oforal 5- All planned timepoints in azacytidine and each Cycles 1, 2, and3 combination agent, including observed maximum concentration (Cmax),time at which maximum concentration is observed (tmax), half-life (t½),area under the curve (AUC), and other parameters as appropriateExploratory Pharmacodynamics To evaluate the Pd of each Time fromachieving first (Pd) combination agent to associate MRD response to lastMRD drug exposure with extent of negative assessment, having a targetmodulation and/or positive MRD result or efficacy/tolerability (in eachincrease in disease burden by treatment cohort during at least one log.Death will be dose/schedule escalation to censored. RP2D) PK/Pd Assessthe relationship between All planned timepoints in PK/Pd biomarkers andclinical Cycles 1, 2, and 3 outcomes of oral 5-azacytidine incombination with venetoclax Correlative Evaluation of molecular,cellular Approximately 8 months Sciences and metabolic markers inperipheral blood (PB) and/or bone marrow (BM) which may be predictive ofantitumor activity and/or resistance. Utilizing both pre- and post-treatment bone marrow biopsy samples acquired for purposes of diseaseassessment, change in baseline of candidate biomarkers will also beassessed using molecular/cell based assays ie, multi-parametric flowcytometry, DNA (gene sequencing), RNA expression (RNAseq), and/or in exvivo assays. For example (but not limited to), MRD: MFC or NGS/PCR basedassays; Gene mutation characterization: using next generation sequencingat baseline, during treatment, to relapse. Oral 5-azacytidine: DNAmethylation changes in whole blood/PBMC/PBLs or BM Venetoclax: BCL-2expression/ amplification levels and characterization of other Bcl-2family members, and/or Bcl-2 mimetic assays Gilteritinib: pFLT3/FLT3,allelic ratio for FLT3, pSTAT5/STAT5 Abbreviations: AE = adverse event;AUC = area under the blood concentration time-curve; BM = bone marrow;Cmax = maximum blood concentration; CR = complete response; CRi =complete response with incomplete recovery of PB counts; CRR = completeresponse rate; CRp = complete response with incomplete plateletrecovery; DLT = dose-limiting toxicity; DOR = duration of response; EFS= event free survival; IP = investigational product; MAD = maximumaccepted dose; MLFS = morphologic leukemia-free state; MTD =maximum-tolerated dose; ORR = overall response rate; OS = overallsurvival; PB = peripheral blood; PCR = polymerase chain reaction; Pd =pharmacodynamics; PD = progressive disease; PK = pharmacokinetics; PR =partial response; T½ = elimination half-life; tmax = time to peak(maximum) blood concentration

Study Design This is an open-label, Phase 1b/2, multi-center,dose-determination, dose expansion, umbrella study that will evaluatethe safety, tolerability, and preliminary efficacy of oral 5-azacytidineas a backbone in combination with biomarker directed and novel therapiesin 2 separate disease cohorts: AML and HR-MDS.

Newly diagnosed patients with AML who are ineligible to receiveintensive induction chemotherapy due to age≥75 years old, or ≥18 yearsold with comorbidities; and AML patients≥18 years old, who relapsed orrefractory to 1 or 2 standard induction treatments will be enrolled inthis study. Newly diagnosed patients with HR-MDS who are ≥18 years oldwill be enrolled in this study.

The study will include 2 parts: Phase 1b dose-finding and Phase 2 doseexpansion.

Phase 1b, dose-finding part will evaluate safety, tolerability,preliminary efficacy, establish the MTD/MAD and determine the RP2D oforal 5-azacytidine in combination with venetoclax or gilteritinib.Dose-finding part will use dose determination rules based on an mTPI-2method. Patients will enroll to one of the following combination arms:

-   -   Cohort 1 AML        -   Arm A: oral 5-azacytidine+Venetoclax for newly diagnosed AML            with all mutations        -   Arm B: oral 5-azacytidine+Gilteritinib for newly diagnosed            or R/R AML with a FLT3 ITD or TKD    -   Cohort 2 HR-MDS        -   Arm C: oral 5-azacytidine+Venetoclax for newly diagnosed,            high and very high-risk MDS patients per IPSS-R

Phase 2, dose expansion part will further evaluate the safety, PK/Pd andefficacy of oral 5-azacytidine in combination with venetoclax at theRP2D. Patients will enroll to one of the following:

-   -   Cohort 1 AML: oral 5-azacytidine+Venetoclax for newly diagnosed        AML with all mutations    -   Cohort 2 HR-MDS: oral 5-azacytidine+Venetoclax for newly        diagnosed HR-MDS per Revised International Prognostic Scoring        System (IPSS-R)

The study consists of a Screening Period, a Treatment Period(dose-finding and dose expansion), and a Follow-up Period. The studydesign is illustrated in FIG. 18.

In one embodiment of this study, additional treatment arms are included,such those described in Example 2. For instance, an additional treatmentarm may include oral 5-azacytidine+Ivosidenib in AML patients with IDH1mutation (Combination Arm A) and/or oral 5-azacytidine+Enasidenib in AMLpatients with IDH2 mutation (Combination Arm B).

Dose-Finding Phase The objective of the dose-finding part of the studyis to identify the MTD/MAD (if reached) and/or RP2D of oral5-azacytidine in combination with venetoclax or gilteritinib in 28-daycycles. The dose-finding part is planned to evaluate 5 dose levels oforal 5-azacytidine in combination with venetoclax or gilteritinib (Table7) and will enroll 3 to 9 patients in each dose level. Approximately 9to 18 patients will enroll into each combination arm.

TABLE 7 Dose Levels by Combination Arms and Disease Cohort AML CohortHR-MDS Cohort Arm A: Arm B: Arm C: Oral 5-Azacytidine + Oral5-Azacytidine + Oral 5-Azacytidine + Venetoclax ^(a) GilteritinibVenetoclax ^(a, c) Dose Level 2 Oral 5-Azacytidine Oral 5-AzacytidineOral 5-Azacytidine 300 mg QD x 21 d + 300 mg QD x 21 d + 300 mg QD x 21d + Venetoclax 400 mg Gilteritinib 120 mg Venetoclax 400 mg QD x 28 d QDx 28 d QD x 14 d Starting Dose Oral 5-Azacytidine Oral 5-AzacytidineOral 5-Azacytidine (Dose Level 1) 300 mg QD x 14 d + 300 mg QD x 14 d +300 mg QD x 14 d + Venetoclax 400 mg Gilteritinib 120 mg Venetoclax 400mg QD x 28 d QD x 28 d QD x 14 d Dose Level −1A Oral 5-Azacytidine Oral5-Azacytidine Oral 5-Azacytidine 200 mg QD x 14 d + 200 mg QD x 14 d +200 mg QD x 14 d + Venetoclax 400 mg Gilteritinib 120 mg Venetoclax 400mg QD x 28 d QD x 28 d QD x 14 d Dose Level −1B Oral 5-Azacytidine Oral5-Azacytidine Oral 5-Azacytidine 300 mg QD x 14 d + 300 mg QD x 14 d +300 mg QD x 14 d + Venetoclax 400 mg Gilteritinib 80 mg Venetoclax 200mg QD x 21 d ^(b) QD x 28 d QD x 14 d Dose Level −2 Oral 5-AzacytidineOral 5-Azacytidine Oral 5-Azacytidine 200 mg QD x 14 d + 200 mg QD x 14d + 200 mg QD x 14 d + Venetoclax 400 mg Gilteritinib 80 mg Venetoclax200 mg QD x 21 d ^(b) QD x 28 d QD x 14 d ^(a) For Cycle 1 only,venetoclax will be administered using a ramp-up dosing schedule: 100 mgon Day 1, 200 mg on Day 2, 400 mg on Day 3 and beyond ^(b) For Arm Adose levels −1B and −2, venetoclax will decrease by treatment durationin days to 400 mg x 21 days instead of 28 days. ^(c) For Arm E,venetoclax dosing will be 400 mg QD x 14 days for dose levels 1, 2, and−1A. The venetoclax de-escalated dose will be 200 mg QD x 14 days fordose levels −1B and −2,

In each dose level, dose limiting toxicities (DLTs) will be assessed forthe respective DLT period, defined as the first 28 days followinginitiation of treatment with oral 5-azacytidine (Cycle 1). Apre-calculated decision table of all the optimal decisions will be usedto guide the selection of doses and/or schedules. For a dose level to befor dose expansion, at least 6 patients should be evaluated for DLTduring the dose escalation to declare a tolerable dose level.

For a patient to be considered DLT evaluable, the patient must haveeither a Cycle 1 DLT after receiving at least one dose of oral5-azacytidine or completed Cycle 1 without a DLT. If a patient is takenoff study for reasons other than toxicities (ie, personal reason ordisease progression) prior to completing 80% of the planned doses oforal 5-azacytidine (11/14 or 16/21 days of 28-day cycle) and thecombination agent (11/14, 16/21, or 22/28 days for venetoclax; or 22/28days for gilteritinib) of the first cycle of therapy, this patient willnot be considered as having completed the treatment cycle and will bereplaced.

After the evaluation of the 5 planned dose levels of oral 5-azacytidinewith venetoclax or gilteritinib is complete, the MTD and/or RP2D will bedetermined, and the dose expansion part will open.

During the dose-finding part, the Investigators will recommend doseescalation/de-escalation decisions and the combination RP2D of oral5-azacytidine for use in the dose expansion based on an integratedassessment of the safety, PK and PD data, and preliminary efficacyinformation. Dose determination decision rules will be guided by anmTPI-2 design (Guo et al., Contemp Clin Trials, 2017 July: 58:23-33),and a target DLT rate of 30% will be used to make recommendations to theSRC for the dose level of the next enrolled patient.

Dose Expansion Phase Once the MTD and/or RP2D of oral 5-azacytidine andvenetoclax combination therapy has been determined, approximately 61newly diagnosed AML, and 54 newly diagnosed MDS patients will beenrolled in two separate disease cohorts. The dose expansion part willfurther evaluate safety and efficacy of the administered combinationRP2D of oral 5-azacytidine with venetoclax.

After dose determination is complete for each combination arm, allongoing patients will continue to receive the same dose and treatmentuntil discontinuation.

Dose reductions may occur based on the observed safety per the dosemodification guidelines

Study Duration The expected duration of the entire study isapproximately 4 years, which includes an overall enrollment period ofapproximately 24 months (12 months in Phase 1b, 12 months in Phase 2),and approximately 2 years duration per patient: a 28-day ScreeningPeriod, a Treatment Period of approximately 12 months, and a Follow-upPeriod of 1 year post last dose. The actual duration of the study willbe dependent upon the median treatment duration and follow-up forpatients.

End of Trial The End of Trial is defined as either the date of the lastvisit of the last patient to complete the post-treatment follow-up, orthe date of receipt of the last data point from the last patient that isrequired for primary, secondary and/or exploratory analysis, aspre-specified in the protocol, whichever is the later date.

Study Population/Estimated No. Patients Approximately 169 patients areplanned for enrollment. The Phase 1b dose-finding part will enroll up toapproximately 54 patients (36 AML, 18 MDS). Each combination arm toenroll approximately 18 patients to evaluate safety, tolerability andpreliminary efficacy. The Phase 2 dose expansion part will enrollapproximately 115 patients (61 AML, 54 MDS).

Key Inclusion Criteria Patients must satisfy the following criteria tobe enrolled in the study:

-   -   1. Patient is ≥18 years of age at the time of signing the        informed consent form (ICF).    -   2. AML Cohort: confirmation of the following for AML as defined        by the updated 2016 World Health Organization (WHO)        Classification.        -   a. First relapse (ie, recurring after having achieved an            initial response [CR/CRi/CRp] to intensive induction            chemotherapy except acute promyelocytic leukemia [APML]); or        -   b. Persisting/refractory after 1 to 2 intensive induction            courses (ie, no response after 1 to 2 prior chemotherapy            regimens); or        -   c. Newly diagnosed AML who are not candidates for intensive            induction chemotherapy due to age≥75 years old, or ≥18 years            old with any of the following comorbidities:            -   Eastern Cooperative Oncology Group (ECOG) performance                status of 2            -   Cardiac history of Congestive Heart Failure (CHF)                requiring treatment or Ejection Fraction≤50% or chronic                stable angina determined by multi-gated acquisition                (MUGA) or echocardiogram (ECHO)            -   Creatinine clearance≥30 mL/min to <45 mL/min calculated                by Cockcroft-Gault formula            -   Moderate hepatic impairment with total bilirubin>1.5 to                ≤3.0× upper limit of normal (ULN)            -   Any other comorbidity that the investigator judges to be                incompatible with intensive chemotherapy must be                reviewed by the Sponsor during screening and before                study enrollment.        -   d. Intermediate or poor risk status cytogenetics for newly            diagnosed AML (Appendix E)        -   e. Confirmed AML specific mutation in bone marrow aspirate            and/or peripheral blood sample documented since last            progression/relapse (or recent testing for newly diagnosed)            as detected by an approved Institutional lab test:            -   Arm A: all mutations            -   Arm B: FLT3 (ITD or TKD)    -   3. MDS Cohort:        -   f. Confirmation of diagnosis of previously untreated primary            or secondary MDS as defined by the WHO Classification.            Results of pathology review are required prior to receiving            the first dose of IP.        -   g. Confirmation of the MDS risk classification per IPSS-R            High or Very High risk. Results of pathology review are            required prior to receiving the first dose of IP.    -   4. Eastern Cooperative Oncology Group (ECOG) performance status        of 0, 1 or 2    -   5. Patients must have the following baseline laboratory values:        -   h. White blood cell (WBC) count of ≤25×10⁹/L. Hydroxyurea or            leukapheresis are permitted to meet this criterion. Note:            hydroxyurea is allowed up to 24 hours prior to the starting            treatment and limited use only during Cycle 1 for            cytoreduction (to control high white blood cell count).        -   i. Potassium and magnesium within normal limits or            correctable with supplements.        -   j. Uric acid≤7.5 mg/dL (446 μmol/L). Prior and/or concurrent            treatment with hypouricemic agents (eg, allopurinol,            rasburicase) are allowed. Rasburicase is contraindicated in            patients with baseline glucose-6-phosphate dehydrogenase            (G6PD) deficiency.        -   k. International normalized ratio (INR)<1.5×ULN and            activated partial thromboplastin time (aPTT)<1.5×ULN.    -   6. Adequate organ function defined as:        -   l. Renal function: Creatinine clearance≥30 mL/minute,            calculated by the Cockcroft-Gault formula or measured by 24            hours urine collection        -   m. Hepatic function: aspartate aminotransferase (AST),            ALT≤3.0×ULN, bilirubin≤1.5×ULN, unless due to Gilbert's            syndrome or leukemic organ involvement. Patients who are <75            years of age may have a bilirubin of ≤3.0×ULN.        -   n. Left ventricular ejection fraction (LVEF)>40% by            multigated acquisition (MUGA) or (echocardiogram) ECHO (see            exclusion criteria #22 for any additional LVEF requirement            in each combination arm)    -   7. Agree to serial bone marrow aspirate/biopsies    -   8. Females of childbearing potential (FCBP)* must:        -   o. Have two negative pregnancy tests as verified by the            Investigator prior to starting study therapy. She must agree            to ongoing pregnancy testing during the course of the study,            and after end of study therapy. This applies even if the            patient practices true abstinence from heterosexual contact.        -   p. Either commit to true abstinence from heterosexual            contact (which must be reviewed on a monthly basis and            source documented) or agree to use, and be able to comply            with effective contraception without interruption during the            study therapy (including dose interruptions), and for 90            days after discontinuation of study therapy, or longer if            required for each compound and/or by local regulations.        -   q. For Combinations Arm A and Arm C: the timeframe is            extended to 6 months following the last study treatment            dose.    -   9. Male patients must practice true abstinence* (which must be        reviewed on a monthly basis) or agree to use a condom during        sexual contact with a pregnant female or a female of        childbearing potential while participating in the study, during        dose interruptions and for at least 90 days following        investigational product discontinuation, or longer if required        for each compound and/or by local regulations, even if he has        undergone a successful vasectomy.        -   r. For Combinations Arm A and Arm C: the timeframe is            extended to 6 months following the last study treatment            dose.    -   10. Patients must understand and voluntarily sign an ICF prior        to any study-related assessments/procedures being conducted.    -   11. Patients is willing and able to adhere to the study visit        schedule and other protocol requirements.

Exclusion Criteria The presence of any of the following will exclude apatient from enrollment:

-   -   1. Patient is suspected or proven to have acute promyelocytic        leukemia (APML) based on morphology, immunophenotype, molecular        assay, or karyotype.    -   2. Patient has AML secondary to chronic myelogenous leukemia or        patient is BCR-ABL1 t(9; 22)(q34;q11) positive.    -   3. Patient has received systemic anticancer therapy or        radiotherapy<28 days prior to the start of study treatment. Note        that hydroxyurea is allowed prior to the start of study        treatment for the control of leukocytosis.    -   4. Patient has received therapy with hypomethylating agent (HMA)        for AML or greater than 2 cycles of HMA for MDS    -   5. Patient has received investigational agents<28 days or 5        half-lives, whichever is longer, prior to the start of study        treatment    -   6. Patient has undergone hematopoietic stem cell transplant        (HSCT) within 90 days prior to the start of study treatment, or        on immunosuppressive therapy post HSCT at the time of screening,        or with graft-versus-host disease (GVHD). The use of a stable        dose of oral steroid post-HSCT, or topical steroids for ongoing        skin GVHD is permitted.    -   7. Patient has persistent, clinically significant        non-hematologic toxicities from prior therapies which have not        recovered to <Grade 2    -   8. Patient has or is suspected of having central nervous system        (CNS) leukemia. Evaluation of cerebrospinal fluid is only        required if CNS involvement by leukemia is suspected during        screening.    -   9. Patient has an active, uncontrolled systemic fungal,        bacterial, or viral infection (defined as ongoing signs/symptoms        related to the infection without improvement despite appropriate        antibiotics, antiviral therapy, and/or other treatment). The        patient should be afebrile for at least 72 hours.    -   10. Patient has immediate life-threatening, severe complications        of leukemia such as uncontrolled bleeding, pneumonia with        hypoxia or shock, and/or disseminated intravascular coagulation    -   11. Patient has prior history of malignancy, other than MDS, MPN        or AML, unless the patient has been free of the disease for ≥1        year prior to the start of study treatment. However, patients        with the following history/concurrent conditions are allowed:        -   Basal or squamous cell carcinoma of the skin        -   Carcinoma in situ of the cervix        -   Carcinoma in situ of the breast        -   Incidental histologic finding of prostate cancer (T1a or T1b            using the tumor, nodes, metastasis clinical staging system)    -   12. Patient is known seropositive or active infection with human        immunodeficiency virus (HIV), or active infection with hepatitis        B virus (HBV) or hepatitis C virus (HCV)    -   13. Patient is known to have dysphagia, short-gut syndrome,        gastroparesis, or other conditions that limit the ingestion or        gastrointestinal absorption of drugs administered orally    -   14. Patients has uncontrolled hypertension (systolic blood        pressure [BP]>180 mmHg or diastolic BP>100 mmHg) or has not been        stable for at least 1 month prior to treatment    -   15. Significant active cardiac disease within the previous 6        months prior to signing the ICF, including:        -   New York Heart Association (NYHA) Class III or IV congestive            heart failure (see Appendix J)        -   Unstable angina or angina requiring surgical or medical            intervention;        -   Significant cardiac arrhythmia; and/or        -   Myocardial infarction    -   16. Patient is a pregnant or lactating female    -   17. Patient has known or suspected to have hypersensitivity to        any of the components of the assigned study treatments    -   18. Patient has any significant medical condition, laboratory        abnormality, or psychiatric illness that would prevent the        patient from participating in the study    -   19. Patient has any condition including the presence of        laboratory abnormalities, which places the patient at        unacceptable risk if he/she were to participate in the study    -   20. Any investigational therapy within 28 days prior to        initiation study treatment    -   21. Patient has any condition that confounds the ability to        interpret data from the study    -   22. Additional exclusion criteria for patients based on planned        Combination Arm are:        -   Combination Arm A (AML all mutations) and Arm C (HR-MDS):            -   Received strong CYP3A inhibitors, moderate CYP3A                inhibitors, strong CYP3A inducers, moderate CYP3A                inducers, strong CYP2C8 inhibitors, CYP2C8 substrates,                or OATP1B1/3 substrates within 7 days prior to                initiation of study treatment.            -   Received strong CYP2C8 inducers within 14 days prior to                initiation of study treatment            -   Received live attenuated vaccines prior to initiation of                study treatment.        -   Combination Arm B (non-specific FLT3):            -   Patient has heart rate corrected QT interval (QTc)                interval (ie, Fridericia's correction [QTcF])≥450 ms or                other factors that increase the risk of QT prolongation                or arrhythmic events (eg, heart failure, hypokalemia,                family history of long QT interval syndrome) at                screening            -   Patient is taking those medications (list of prohibited                medications will be provided in the protocol) that are                known to prolong QT interval unless the patient can be                transferred to other medications at least 5 half-lives                prior to the start of study treatment            -   Patient has a history or presence of sustained                ventricular tachycardia; any history of ventricular                fibrillation or torsades de pointes; bradycardia defined                as heart rate (HR)<50 bpm; right bundle branch                block+left anterior hemiblock (bifascicular block); an                ejection fraction≤45% assessed by MUGA or ECHO within 14                days of cycle 1 day 1            -   Patient is taking medications which are P-glycoprotein                (P-gp) substrates and strong CYP3A4 inhibitors, and                inducers within the previous 7 days.            -   Patients with hypokalemia and hypomagnesemia at                Screening (values below lower limit of normal [LLN]).            -   Patients with Long QT Syndrome at Screening.            -   Patient requires treatment with concomitant drugs that                are strong inhibitors or inducers of P-gp with the                exception of drugs that are considered absolutely                essential for the care of the patient.            -   Patient requires treatment with concomitant drugs that                target serotonin 5-hydroxytryptamine receptor 1 (5HT1R)                or 5-hydroxytryptamine receptor 2B (5HT2BR) or sigma                nonspecific receptor with the exception of drugs that                are considered absolutely essential for the care of the                patient.    -   23. Patient has any significant medical condition, laboratory        abnormality, or psychiatric illness that would prevent the        patient from participating in the study.    -   24. Patient has any condition including the presence of        laboratory abnormalities, which places the patient at        unacceptable risk if he/she were to participate in the study.    -   25. Patient has any condition that confounds the ability to        interpret data from the study.

Treatment Period The patient may be enrolled once allinclusion/exclusion criteria are verified and the patient is deemed tobe eligible. The patient must start treatment within 3 days ofenrolment. For all subsequent visits, an administrative window of ±3days is permitted.

Prior to first venetoclax dose (ie, before ramp-up), patients willreceive prophylactic measures including adequate hydration andanti-hyperuricemic agents which will be continued during the ramp-upphase (ie, before ramp-up and continue until after final dose is reachedor longer as clinically indicated). Treatment cycles are 28 days induration.

Investigational Products

The study will use oral 5-azacytidine as 200 mg and 300 mg tablets fororal administration. Each tablet is formulated using excipients that aregenerally regarded as safe and are used in marketed drug products.

The marketed form of venetoclax (VENCLEXTA®) will be used in 10 mg, 50mg, and 100 mg tablets for oral administration.

The marketed form of gilteritinib (XOSPATA®) will be used in 40 mgtablets for oral administration.

Treatment Administration and Schedule For dose-finding (Phase 1b),following screening, eligible patients will be assigned to receive5-azacytidine 300 mg orally (PO) once daily on Days 1-14 (or 21) withvenetoclax or gilteritinib in a 28-day treatment cycle as described inthis example.

In order to allow for the best opportunity to benefit from thetreatment, and given the mechanism of action of oral 5-azacytidine andthe median time to response for the venetoclax or gilteritinibcombination, patients should be treated for at least 3 cycles withvenetoclax or at least 6 cycles with gilteritinib, although patientswill be discontinued from the treatment sooner if they demonstratedocumented relapse from CR or PR, disease progression, unacceptable AEs,intercurrent illness that prevents further administration of treatment,Investigator's decision to withdraw the patient, withdrawal of consent,noncompliance with trial treatment or procedure requirements, death, oradministrative reasons.

Oral 5-azacytidine will be given PO once daily for 14 (or 21) days in a28-day cycle on Days 1-14 (or 21). Oral 5-azacytidine will beadministered about the same time each day with approximately 240 mL (8ounces) of room temperature water, with or without food.

In Phase 1b dose-finding part of the study, starting dose level of oral5-azacytidine will be 300 mg×14 days PO QD per 28-day cycle on Days 1-14(or 21). Depending on the number of DLTs, the dose may decrease dose to200 mg×14 days; or if tolerated, dose may stay the same or increasetreatment duration in days to 300 mg×21 days.

When oral 5-azacytidine, venetoclax or gilteritinib will be administeredon the same day, oral 5-azacytidine will be administered first followedby venetoclax or gilteritinib.

Venetoclax will be administered according to the approved label(Venclexta®, 2019) orally QD on Days 1 to 28 per 28-day cycle for AML,patients. Venetoclax dosing for HR-MDS is on Days 1 to 14 per 28-daycycle A brief dose ramp-up occurs for Cycle 1 with a starting dosing of100 mg on Day 1, 200 mg on Day 2, and 400 mg on Day 3 and beyond.Venetoclax will be administered at 400 mg on subsequent days.

In Phase 1b dose-finding part of the study, venetoclax starting doselevel will be 400 mg PO QD×28 days for AML, patients (Arm A) and 400 mgPO QD×14 days for MDS patients (Arm C) in a 28-day cycle. Depending onthe number of DLTs experienced and if related to venetoclax, thevenetoclax dose may de-escalate by decreasing treatment duration to 400mg PO QD×21 days (Arm A), or by decreasing dosage to 200 mg PO QD×14days (Arm C) in a 28-day cycle. In Phase 2 dose expansion, the RP2D willbe given to patients. In Phase 2 dose expansion part, the RP2D ofvenetoclax will continue the same schedule and timing in a 28-day cycle(ie, Cycle 3 is 28 days after Cycle 2, etc).

Patients will be hospitalized during the venetoclax dose ramp-up inCycle 1 for a minimum of 8 days from Day 1 to Day 8. Thishospitalization is required per protocol and does not constitute aserious adverse event. Patients should be instructed to take their dailydose at approximately the same time each day+8 hours.

Gilteritinib (XOSPATA®) will be administered at the recommended startingdose of 120 mg administered orally once a day (QD) on Days 1-28 of each28-day cycle. Patients should be instructed to take their daily dose atapproximately the same time each day.

Safety Follow-up All patients will be followed for 28 days after thelast dose of investigational product (IP) or EOT Visit, whichever occurslater, for AE reporting and concomitant medication information as wellas new disease therapies. The 28-day (±3 days) safety follow-up contactmay be by telephone for non-FCBP. For FCBP, the 28-day safety follow-upwill be completed in the clinic for pregnancy testing requirements. Inaddition, any SAEs made known to the Investigator at any time thereafterthat are suspected of being related to IP.

Survival Follow-up After the end of treatment visit, all patients willbe followed every 4 weeks for survival follow-up for up to 1 year postlast dose or until death, lost to follow-up, or the End of Trialwhichever occurs first. New disease therapies for AML or MDS should becollected at the same time schedule.

Efficacy Assessment Serial blood and bone marrow BM sampling will beused to determine response to study drug therapy starting at Cycle 2 Day1.

At baseline, a bone marrow aspirate (BMA) sample is required. A biopsymust be collected if the aspirate is not available and may be collectedin addition to the aspirate per institutional practice. Cytogenetic andmolecular profiling from the BMA are also required at Screening (unlessthey are available to enter from the patient's medical records from thepast 90 days). Complete blood counts, peripheral blood smear (PBS), andBMAs will be used to determine response to therapy at specifictimepoints. Samples may be obtained up to 4 days prior to the end of thecycle, ie, Days 25 to 28.

Response to treatment will be assessed by the Investigator using locallaboratory results according to the following guidelines per diseasecriteria based on reported hematology laboratory parameters, peripheralblood smear (PBS), bone marrow aspirates (BMAs), and/or biopsies:

-   -   AML treatment will be assessed according to modified European        Leukemia Net (ELN) AML Response Criteria, ie, modified to        include Complete remission with partial hematologic recovery        (CRh) and hematologic response (Döhner, H et al., Blood, 2017        Jan. 26, 129(4): 424-47); and IWG for AML Response Criteria        (Cheson B. D. et al., J Clin Oncol. 2003, 21(24): 4642-92003).        Hematologic improvement (HI) will also be assessed according to        the IWG MDS HI criteria (Cheson B. D. et al., Blood 2006;        108(2): 419-425).    -   MDS treatment will be assessed according to IWG for MDS Response        Criteria (Cheson B. D. et al., Blood 2006; 108(2): 419-425).

Hematologic response will be evaluated as patients with antecedenthematologic disorders may be enrolled onto study. Transfusion dependenceis defined as having received ≥2 units of RBCs and/or platelets within 8weeks prior to study treatment. Transfusion independence is defined as aperiod of 8 weeks with no transfusions.

For AML, progressive disease will be defined as one of the followingchanges from baseline:

-   -   For patients with 5 to 70% bone marrow (BM) blasts at baseline:        a >50% increase of BM blast count percentage from baseline to        ≥20%; or    -   For patients with >70% BM blasts at baseline: a doubling of        absolute blast count in peripheral blood from baseline to        ≥10×10⁹/L (10,000/μL); or    -   New extramedullary disease since last response assessment    -   Progression/relapse after a hematological improvement with at        least one of the following:        -   At least 50% decrement from maximum response levels in            granulocytes or platelets        -   Reduction in Hgb by ≥1.5 g/dL        -   Transfusion dependence

For HR MDS, progressive disease will be defined as one of the followingchanges from baseline:

-   -   Less than 5% blasts: ≥50% increase in blasts to >5% blasts    -   5%-10% blasts: ≥50% increase in blasts to >10% blasts    -   10%-20% blasts: ≥50% increase in blasts to >20% blasts    -   20%-30% blasts: ≥50% increase in blasts to >30% blasts

Any of the following:

-   -   At least 50% decrement from maximum remission/response levels in        granulocytes or platelets    -   Reduction in Hgb concentration by ≥2 g/dL    -   Transfusion dependence

Progressive disease is to be confirmed by 2 consecutive responseassessments separated by at least 1 month. The date of progressivedisease is defined as the first date that one of the conditions abovewas met. In the absence of progressive disease (as defined above) orunacceptable toxicity, patients may continue treatment if they arederiving benefit, as judged by the Investigator.

The marrow aspiration and core sampling (biopsy) should be performedaccording to the standard of care and analyzed at the local site'slaboratory in accordance with the International Council forStandardization in Hematology (ICSH) Guidelines (Lee, J. H. et al., JClin Oncol. 2017, 35 (24): 2754-2763).

Patients are to undergo end-of treatment evaluations when studytreatment is discontinued. The reason for treatment discontinuation willbe recorded in the eCRF pages and in the source document. Patients whodiscontinue study treatment prior to relapse or progression willcomplete site visits. For patients who have discontinued study treatmentdue to relapse or progression, Survival Follow-up can be performed bysite visits or phone calls. Patients will be followed for up to 1 yearpost last dose, or until they have died, are lost to follow up, orwithdrawal of consent for further data collection within 1 year from thedate of last dose.

For acute myeloid leukemia, response criteria will be summarized by bestoverall response categories:

-   -   CR/CRh rate, and overall response rate ORR. The ORR includes all        responses of complete remission (CR) (ie, CR, CRi CRp),        morphologic leukemia-free state (MLFS), and partial remission        (PR). CRh=Complete remission with partial hematologic recovery.        CRi=CR with incomplete hematologic recovery. CRp=C-reactive        protein.    -   Minimal residual disease (MRD) response rate and MRD conversion        rate will also be assessed as efficacy variables. Minimal        residual disease will be assessed by MFC and/or next generation        sequencing (NGS) at the same time as efficacy assessments and        will be assessed centrally. The site will ensure peripheral        blood and BMA/bone marrow biopsy (BMB) samples are collected and        stored for exploratory testing at the time of each BM        collection.    -   Other measures of clinical activity including overall survival        (OS), event-free survival (EFS), RFS, time to response (CR, CRi,        CRp, PR, MLFS), duration of responses (CR, CRh and ORR),        hematologic improvement rate, and a 1-year survival rate will be        summarized.

For myeloid dysplastic syndrome, response criteria will be summarized bybest overall response categories:

-   -   CR, PR, mCR    -   Other measures of clinical activity including OS, EFS, RFS, time        to response and duration of responses for CR, PR, and mCR,        hematologic improvement rate, and a 1-year survival rate will be        summarized.

The results of the clinical study will show that oral 5-azacytidine incombination with venetoclax or gilteritinib at the maximum tolerateddose (MTD), maximum administered dose (MAD) and/or the recommended Phase2 dose (RP2D) is safe and tolerated in patients with AML or higher-riskMDS, which include: (1) patients with newly diagnosed AML with allmutations treated with oral 5-azacytidine in combination withvenetoclax; (2) patients with newly diagnosed or relapsed and/orrefractory AML with a FLT3 ITD or TKD mutation treated with oral5-azacytidine in combination with gilteritinib; and (3) patients withnewly diagnosed high and very high risk MDS treated with oral5-azacytidine in combination with venetoclax.

Further, the results of the clinical study will also show that oral5-azacytidine when given in combination with venetoclax at the RP2D isefficacious in newly diagnosed patients with AML or higher-risk MDS,which include: (1) patients with newly diagnosed AML with all mutationstreated with oral 5-azacytidine in combination with venetoclax; and (2)patients with newly diagnosed newly diagnosed HR-MDS per RevisedInternational Prognostic Scoring System (IPSS-R) treated with oral5-azacytidine in combination with venetoclax.

The present disclosure has been described in connection with certainembodiments and examples; however, the claimed invention should not belimited to such specific embodiments and examples.

While certain embodiments have been illustrated and described, it shouldbe understood that changes and modifications can be made therein inaccordance with the knowledge of one of ordinary skill in the artwithout departing from the technology in its broader aspects as definedin the following claims.

The embodiments illustratively described herein may suitably bepracticed in the absence of any element or elements, limitation orlimitations, not specifically disclosed herein. Thus, for example, theterms “comprising,” “including,” “containing,” etc., shall be readexpansively and without limitation. Additionally, the terms andexpressions employed herein have been used as terms of description andnot of limitation, and there is no intention in the use of such termsand expressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the claimed technology.Additionally, the phrase “consisting essentially of” will be understoodto include those elements specifically recited and those additionalelements that do not materially affect the basic and novelcharacteristics of the claimed technology. The phrase “consisting of”excludes any element not specified.

The present disclosure is not to be limited in terms of the particularembodiments described in this application. Many modifications andvariations can be made without departing from its spirit and scope, aswill be apparent to those skilled in the art. Functionally equivalentmethods and compositions within the scope of the disclosure, in additionto those enumerated herein, will be apparent to those skilled in the artfrom the foregoing descriptions. Such modifications and variations areintended to fall within the scope of the appended claims. The presentdisclosure is to be limited only by the terms of the appended claims,along with the full scope of equivalents to which such claims areentitled. It is to be understood that this disclosure is not limited toparticular methods, reagents, compounds, or compositions, which can ofcourse vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only andis not intended to be limiting.

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatthe disclosure is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges disclosed herein also encompass any and all possible subrangesand combinations of subranges thereof, inclusive of the endpoints. Anylisted range can be easily recognized as sufficiently describing andenabling the same range being broken down into at least equal halves,thirds, quarters, fifths, tenths, etc. As a non-limiting example, eachrange discussed herein can be readily broken down into a lower third,middle third and upper third, etc. As will also be understood by oneskilled in the art, all language such as “up to,” “at least,” “greaterthan,” “less than,” and the like include the number recited and refer toranges which can be subsequently broken down into subranges as discussedabove. Finally, as will be understood by one skilled in the art, a rangeincludes each individual member.

All publications, patent applications, issued patents, and otherdocuments referred to in this specification are herein incorporated byreference as if each individual publication, patent application, issuedpatent, or other document was specifically and individually indicated tobe incorporated by reference in its entirety. Definitions that arecontained in text incorporated by reference are excluded to the extentthat they contradict definitions in this disclosure.

Other embodiments are set forth in the following claims.

1. A method of treating a human having acute myeloid leukemia and/ormyelodysplastic syndrome, wherein the method comprises administering tothe human (i) a pharmaceutical composition comprising 5-azacytidineadministered orally, and (ii) at least one additional therapeutic agent.2. The method of claim 1, wherein the at least one additionaltherapeutic agent comprises an FMS-like tyrosine kinase 3 (FLT3)inhibitor, an isocitrate dehydrogenase 2 (IDH2) inhibitor, an isocitratedehydrogenase 2 (IDH1) inhibitor, and/or a B-cell lymphoma 2 (BCL2)inhibitor.
 3. The method of claim 2, wherein the at least one additionaltherapeutic agent comprises gilteritinib, midostaurin, quizartinib,enasidenib, ivosidenib, and/or venetoclax.
 4. The method of claim 3,wherein the at least one additional therapeutic agent is gilteritinib ormidostaurin.
 5. The method of claim 3, wherein the at least oneadditional therapeutic agent is venetoclax.
 6. The method of claim 1,wherein: (a) the 5-azacytidine and the at least one additionaltherapeutic agent are administered concomitantly; or (b) the5-azacytidine and the at least one additional therapeutic agent areadministered sequentially wherein the 5-azacytidine is administeredfirst.
 7. The method of claim 1, wherein: (a) the 5-azacytidine and theat least one additional therapeutic agent are co-formulated as a singleunit dosage form; or (b) the 5-azacytidine and the at least oneadditional therapeutic agent are formulated as separate dosage forms. 8.The method of claim 1, wherein: (a) the at least one additionaltherapeutic agent is administered parenterally; or (b) the at least oneadditional therapeutic agent is administered orally.
 9. The method ofclaim 1, wherein the 5-azacytidine is administered: (a) at a dose ofabout 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, about250 mg, about 260 mg, about 270 mg, about 280 mg, about 290 mg, about300 mg, about 310 mg, about 320 mg, about 330 mg, about 340 mg, about350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, about400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg, about450 mg, about 460 mg, about 470 mg, about 480 mg, about 490 mg, about500 mg, about 510 mg, about 520 mg, about 530 mg, about 540 mg, about550 mg, about 560 mg, about 570 mg, about 580 mg, about 590 mg, or about600 mg orally; and/or (b) at a dose of about 200 mg; and/or (c) at adose of about 300 mg; and/or (d) for the first seven, fourteen, ortwenty-one days of a 28-day cycle; and/or (e) to the human one or twotimes per day; and/or; (f) in the form of a capsule or a tablet.
 10. Themethod of claim 9, wherein the 5-azacytidine is administered in the formof a non-enteric-coated tablet.
 11. The method of claim 9, wherein the5-azacytidine is administered in the form of an immediate release oralcomposition.
 12. The method of claim 1, wherein the 5-azacytidine isadministered: (a) at a dose of about 200 mg per day for 14 days in a28-day cycle; (b) at a dose of about 300 mg per day for 14 days in a28-day cycle; (c) at a dose of about 200 mg per day for 21 days in a28-day cycle; (d) at a dose of about 300 mg per day for 21 days in a28-day cycle; (e) at a dose of about 200 mg per day for 7 days in a28-day cycle; or (f) at a dose of about 300 mg per day for 7 days in a28-day cycle.
 13. The method of claim 1, wherein the 5-azacytidine isadministered: (a) daily for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, or greater than 14 days, optionally followed by a treatment dosingholiday of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or greaterthan 14 days; (b) daily for 14 or more days, optionally followed by atreatment dosing holiday of 7 or more days; (c) for 21 or more days,optionally followed by a treatment dosing holiday of 7 or more days; (d)for 14 days, optionally followed by a treatment dosing holiday of 14days; (e) for 21 or more days, followed by a treatment dosing holiday of7 or more days; or (f) for 14 days, followed by a treatment dosingholiday of 14 days.
 14. The method of claim 13, wherein at least one ofsteps (a), (b), (c), (d), (e), or (f) are repeated.
 15. The method ofclaim 1, wherein the 5-azacytidine is administered: (a) at a dose ofabout 300 mg daily for 14 days, followed by a treatment dosing holidayof 14 days; (b) at a dose of about 200 mg daily for 14 days, followed bya treatment dosing holiday of 14 days; (c) at a dose of about 300 mgdaily for 21 days, followed by a treatment dosing holiday of 7 days; (d)at a dose of about 200 mg daily, followed by a treatment dosing holidayof 7 days; (e) at a dose of 300 mg daily for 14 days, followed by atreatment dosing holiday of 14 days; (f) at a dose of 200 mg daily for14 days, followed by a treatment dosing holiday of 14 days; (g) at adose of 300 mg daily for 21 days, followed by a treatment dosing holidayof 7 days; or (h) at a dose of 200 mg daily, followed by a treatmentdosing holiday of 7 days.
 16. The method of claim 15, wherein at leastone of steps (a), (b), (c), (d), (e), (f), (g), or (h) are repeated. 17.The method of claim 1, wherein the 5-azacytidine is administered: (a)using a treatment cycle comprising administration of 5-azacytidine perday for 7 days in a 28-day cycle; (b) using a treatment cycle comprisingadministration of 5-azacytidine per day for 14 days in a 28-day cycle;or (c) using a treatment cycle comprising administration of5-azacytidine per day for 21 days in a 28-day cycle.
 18. The method ofclaim 4, wherein the 5-azacytidine and the at least one additionaltherapeutic agent provides a synergistic effect to treat the acutemyeloid leukemia or myelodysplastic syndrome.
 19. The method of claim 4,wherein: (a) the acute myeloid leukemia is characterized as caused by aFMS-like tyrosine kinase-3 internal tandem duplication (FLT3-ITD)mutation; and/or (b) the 5-azacytidine is administered before the atleast one additional therapeutic agent; and/or (c) the 5-azacytidine andat least one additional therapeutic agent augments myeloid cell leukemia1 (MCL-1) degradation; and/or (d) the 5-azacytidine and at least oneadditional therapeutic agent increases median survival as compared to5-azacytidine administered intravenously or subcutaneously and at leastone additional therapeutic agent; and/or (e) the 5-azacytidine and atleast one additional therapeutic agent increases median survival ascompared to 5-azacytidine administered intravenously or subcutaneouslyand at least one additional therapeutic agent by about 10%, about 15%,about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%,about 85%, about 90%, about 95%, or about 100%.
 20. The method of claim5, wherein the 5-azacytidine and the at least one additional therapeuticagent provides a synergistic effect to treat the acute myeloid leukemiaor myelodysplastic syndrome.
 21. The method of claim 5, wherein: (a) theacute myeloid leukemia is characterized as caused by a FLT3-ITDmutation; and/or (b) the acute myeloid leukemia is resistant totreatment with the at least one additional therapeutic agent alone;and/or (c) the 5-azacytidine is administered before the at least oneadditional therapeutic agent; and/or (d) the acute myeloid leukemia isresponsive to treatment with a FMS-like tyrosine kinase-3 (FLT3inhibitor); and/or (e) the acute myeloid leukemia is characterized ashaving an overexpression of MCL-1; and/or (f) the 5-azacytidine primesthe cancer cells for apoptosis mediated by the at least one additionaltherapeutic agent by downregulating the expression of MCL-1; and/or (g)the 5-azacytidine and at least one additional therapeutic agent augmentsMCL-1 degradation; and/or (h) the 5-azacytidine alters cell metabolism;and/or (i) the 5-azacytidine causes cell cycle arrest; and/or (j) the5-azacytidine suppresses oxidative phosphorylation; and/or (k) the5-azacytidine increases expression of activating transcription factor 3(ATF3); and/or (l) the 5-azacytidine decreases expression ofstearoyl-CoA desaturase (SCD); and/or (m) the 5-azacytidine and at leastone additional therapeutic agent increases median survival as comparedto 5-azacytidine administered intravenously or subcutaneously and atleast one additional therapeutic agent; and/or (n) the 5-azacytidine andat least one additional therapeutic agent increases median survival ascompared to 5-azacytidine administered intravenously or subcutaneouslyand at least one additional therapeutic agent by about 10%, about 15%,about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%,about 85%, about 90%, about 95%, or about 100%.
 22. The method of claim1, wherein the method comprises: (a) administering the 5-azacytidinedaily to the human for 1, 2, or 3 days; (b) administering the at leastone additional therapeutic agent to the human for one or more days; and(c) optionally repeating steps (a) and (b).
 23. The method of claim 1,wherein the method comprises: (a) administering the 5-azacytidine dailyto the human for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days;(b) administering the at least one additional therapeutic agent to thehuman for one or more days; and (c) optionally repeating steps (a) and(b).
 24. The method of claim 1, wherein the method comprises: (a)administering the 5-azacytidine daily to the human for 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days; (b)administering the at least one additional therapeutic agent to the humanfor one or more days; and (c) optionally repeating steps (a) and (b).25. The method of claim 1, wherein the method comprises: (a)administering the 5-azacytidine daily to the human for 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, or 14 days of a 28-day cycle; (b) concurrentlyadministering the at least one therapeutic agent daily to the human for1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, or 28 days of a 28-day cycle; and (c)optionally repeating steps (a) and (b).
 26. The method of claim 25,wherein administering the at least one additional therapeutic agentcomprises administering gilteritinib, midostaurin, quizartinib,enasidenib, ivosidenib, and/or venetoclax.
 27. The method of claim 1,wherein the method comprises: (a) administering the 5-azacytidine dailyto the human for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, or 21 days of a 28-day cycle; (b) concurrentlyadministering the at least one additional therapeutic agent daily to thehuman for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days of a 28-day cycle; and(c) optionally repeating steps (a) and (b).
 28. The method of claim 27,wherein administering the at least one additional therapeutic agentcomprises administering gilteritinib, midostaurin, quizartinib,enasidenib, ivosidenib, and/or venetoclax.
 29. The method of claim 1,wherein the method comprises the sequential steps of: (a) administeringthe 5-azacytidine to the human for 7 days of a 28-day cycle; (b)administering the at least one additional therapeutic agent to the humanfor 1 day of a 28-day cycle; (c) administering 5-azacytidine to thehuman for 6 days of a 28-day cycle; and (d) repeating steps (a) to (c)after 7 days of a resting period.
 30. The method of claim 1, wherein themethod comprises the sequential steps of: (a) administering5-azacytidine daily to the human for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days of a 28-day cycle; (b)administering the at least one additional therapeutic agent to the humandaily for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, or 21 days of a 28-day cycle; (c) administering 5-azacytidine tothe human daily for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, or 21 days of a 28-day cycle; and (d) optionallyrepeating steps (a) and (c) after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, or 21 days of a resting period.
 31. Themethod of claim 1, wherein the human has acute myeloid leukemia.
 32. Themethod of claim 1, wherein the human has myelodysplastic syndrome. 33.The method of claim 32, wherein the myelodysplastic syndrome is high andvery high risk myelodysplastic syndrome as defined by the RevisedInternational Prognostic Scoring System (IPSS-R).